A CRISPR-Cas9 Toolbox For Multiplexed Plant Genome Editing and Transcriptional Regulation - 2015

Breakthrough Technologies
A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome

Editing and Transcriptional Regulation1[OPEN]
Levi G. Lowder 2, Dengwei Zhang 2, Nicholas J. Baltes, Joseph W. Paul III, Xu Tang, Xuelian Zheng,
Daniel F. Voytas, Tzung-Fu Hsieh, Yong Zhang*, and Yiping Qi*
Department of Biology, East Carolina University, Greenville, North Carolina 27858 (L.G.L., J.W.P., Y.Q.);
Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and
Technology of China, Chengdu 610054, China (D.Z., X.T., X.Z., Y.Z.); Department of Genetics, Cell Biology,
and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota
55455 (N.J.B., D.F.V.); and Department of Plant and Microbial Biology and Plants for Human Health Institute,
North Carolina State University, North Carolina Research Campus, Kannapolis, North Carolina 28081 (T.-F.H.)
ORCID IDs: 0000-0003-4241-3498 (J.W.P.); 0000-0002-7475-7888 (X.Z.); 0000-0001-7584-3721 (T.-F.H.); 0000-0003-3704-4835 (Y.Z.);
0000-0002-9556-6706 (Y.Q.).
The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular
biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant
systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For
example, signicant plant molecular biology expertise and effort is still required to generate functional expression constructs
that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing,
which is a signicant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and
wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive
molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol
and reagents to quickly and efciently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots
using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing
and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this
toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa),
demonstrating its utility for basic and applied plant research.
Customizable sequence-specic nucleases (SSNs)

are powerful tools for plant genome editing (Voytas,
2013; Carroll, 2014; Puchta and Fauser, 2014). SSNs
can induce sequence-specic DNA double-strand
breaks (DSBs), which are subsequently repaired
by either nonhomologous end joining (NHEJ) or
1
This work was supported by East Carolina University (startup
funds to Y.Q.), the National Science Foundation of China (grant nos.
31271420, 31330017, and 31371682 to Y.Z.), the National Transgenic
Major Project (grant no. 2014ZX0801003B002 to Y.Z.), and the National Science Foundation (grant nos. MCB 0209818, 1051339209,
and DBI 0923827 to D.F.V.).
2
These authors contributed equally to the article.
* Address correspondence to qiy@ecu.edu and zhangyong916@
uestc.edu.cn.
The author responsible for distribution of materials integral to the
ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Yiping Qi (qiy@ecu.edu).
L.G.L., D.F.V., T.-F.H., Y.Z., and Y.Q. designed the experiments;
L.G.L., D.Z., N.J.B., J.W.P., X.T., X.Z., T.-F.H., Y.Z., and Y.Q. conducted
the experiments and analyzed the data; L.G.L., D.F.V., Y.Z., and Y.Q.
wrote the article.
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homologous recombination (Kanaar et al., 1998; Puchta,

2005). By directing DNA DSBs and harnessing DNA
repair pathways, mutations or precise modications
can be introduced within a genome at desired loci.
Historically, meganucleases or zinc nger nucleases
(ZFNs) have been the SSNs of choice, but they are notoriously difcult to engineer and function inconsistently across different genetic loci (Carroll, 2011; Hafez
and Hausner, 2012). As a result, such technologies have
not been widely adopted within the plant research
community (Puchta and Fauser, 2013).
Recent advances in SSN engineering and design
have provided more viable options for plant genome
editing. For ZFNs, new engineering methods have
been developed, namely OPEN and CoDA (Maeder
et al., 2008; Sander et al., 2011). We previously used
OPEN- or CoDA-engineered ZFNs to successfully
target endogenous plant genes, creating mutations, gene
replacements, deletions, or inversions in Arabidopsis
(Arabidopsis thaliana), tobacco (Nicotiana benthamiana),
and soybean (Glycine max; Townsend et al., 2009; Zhang
et al., 2010; Curtin et al., 2011; Qi et al., 2013b, 2014).
However, ZFNs suffer from target site availability, activity, and occasionally toxicity (Carroll, 2011; Reyon
et al., 2011; Sander et al., 2011). As ZFN limitations
Plant Physiology, October 2015, Vol. 169, pp. 971985, www.plantphysiol.org 2015 American Society of Plant Biologists. All Rights Reserved.
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Lowder et al.
surfaced, a more versatile transcription activator-like

effector nuclease (TALEN)-based SSN platform emerged
(Christian et al., 2010; Li et al., 2011; Miller et al., 2011).
Compared to ZFNs, TALENs possess a broader targeting
range and are less difcult to engineer (Bogdanove and
Voytas, 2011; Doyle et al., 2012). Moreover, TALENs appear to be more mutagenic than ZFNs (Chen et al., 2013)
and are highly specic (Juillerat et al., 2014). To promote
TALEN technology, we developed a streamlined TALEN
assembly method (Cermak et al., 2011), which was later
used for successful genome editing in many plant species
(Christian et al., 2013; Shan et al., 2013a; Wendt et al., 2013;
Zhang et al., 2013; Lor et al., 2014; Wang et al., 2014).
Unfortunately, both ZFN and TALEN technologies require engineering of DNA-binding domains for individual targeting applications, demanding signicant effort
and expertise in molecular cloning.
Most recently, the Streptococcus pyogenes clustered
regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated Protein9 (Cas9) system
has burst on the scene, after it was shown to effectively
mediate RNA-guided DNA double-strand breaks in
bacteria and mammalian cells (Jinek et al., 2012; Cong
et al., 2013; Mali et al., 2013b). A distinct feature of
CRISPR/Cas9 is that DNA cleavage sites are recognized through Watson-Crick base pairing by a guide
RNA (gRNA)/Cas9 complex. This feature drastically
simplies DNA targeting. DNA cleavage by CRISPR/
Cas9 requires three components: Cas9 protein,
CRISPR RNA, and trans-activating CRISPR RNA (Jinek
et al., 2012). However, the two RNA components have
been reduced to a single gRNA that can be functionally
expressed under small nuclear RNA promoters such as
U6 or U3 (Jinek et al., 2012; Cong et al., 2013; Mali et al.,
2013b). This improvement further simplies the
CRISPR/Cas9 system and enhances reagent delivery.
After initial reports, many studies quickly announced
successful and effective CRISPR/Cas9-mediated genome editing in plants (Feng et al., 2013, 2014; Jiang
et al., 2013; Li et al., 2013; Mao et al., 2013; Miao et al.,
2013; Nekrasov et al., 2013; Shan et al., 2013b; Xie and
Yang, 2013; Fauser et al., 2014; Schiml et al., 2014; Zhou
et al., 2014). Due to simplied engineering of target
specicity and its dual-component nature, CRISPR/
Cas9 allows for simultaneous targeting of multiple genomic loci. This multiplexing feature has been demonstrated in a number of the aforementioned studies
where two gRNAs were simultaneously expressed
along with Cas9 protein (Li et al., 2013; Schiml et al.,
2014; Zhou et al., 2014).
Another advantage of CRISPR/Cas9 is that it can
readily be converted to a reagent that creates singlestrand breaks (a nickase). Zinc nger nickases and
transcription activator-like effector (TALE) nickases
have been made by generating a D450A mutation in
the FokI nuclease domain, but these nickases appear to
have noticeable residual nuclease activity (Ramirez
et al., 2012; Wang et al., 2012; Wu et al., 2015). On the
contrary, either of the two endonuclease domains
of Cas9, HNH, and RuvC can be mutated to form
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nickases (Gasiunas et al., 2012; Jinek et al., 2012). While

CRISPR/Cas9 may pose a potential risk for off-target
activity compared with ZFNs or TALENs (Fu et al.,
2013), Cas9 nickases could greatly alleviate this
problem. For example, mutations have been effectively generated at specic loci using paired Cas9
nickases with minimal off-target mutagenic effects in
mice (Shen et al., 2014), human cells (Ran et al., 2013),
Drosophila melanogaster (Ren et al., 2014), and Arabidopsis (Schiml et al., 2014). Implementation of paired
nickases requires simultaneous expression of at
least two gRNAs.
The CRISPR/Cas9 system can also be repurposed for
transcriptional regulation. Fusion of a transcriptional
activator domain such as VP16 or VP64 (Sadowski et al.,
1988; Beerli et al., 1998) to a deactivated Cas9 (dCas9)
lacking endonuclease activity can up-regulate endogenous gene expression in human cells (Cheng et al., 2013;
Gilbert et al., 2013; Maeder et al., 2013; Mali et al., 2013a;
Perez-Pinera et al., 2013). In these studies, single gRNAmediated gene activation was generally not highly
effective. However, when multiple gRNAs were
coexpressed, a synergistic or additive transcriptional
activation was observed. Repression of endogenous
genes has also been demonstrated by expression of
dCas9 or dCas9-repressor fusion proteins in human
cells (Gilbert et al., 2013; Qi et al., 2013a) and tobacco
(Piatek et al., 2014). Complex gene expression programs
can be engineered with modied gRNA scaffolds that
can simultaneously induce transcriptional activation
or repression (Zalatan et al., 2015). This would be a
potentially important tool for plant synthetic biology.
With the ability to genetically modify genes and
regulate their transcription, the CRISPR/Cas9 system is
clearly a powerful tool for basic and applied research in
plants. To unleash the full potential of CRISPR/Cas9
for plant-based applications, an easy-to-use multiplexed assembly system is needed. Here, we developed
a toolbox with a streamlined protocol for assembly of
multifaceted multiplexed CRISPR/Cas9 reagents together into transfer DNA (T-DNA) vectors. The assembly is based on efcient Golden Gate cloning and
Gateway recombination methods with no PCR required. By testing the toolbox in dicot and monocot
plants, we demonstrated the exibility of this toolbox
for plant genome and transcriptional regulation.
RESULTS
An Efcient Assembly System for Diverse, Multiplexed
CRISPR/Cas9 Applications
We sought to design a multifaceted and easy-to-use

CRISPR/Cas9 system for the plant research community. As illustrated in Figure 1, potential applications include, but are not limited to (1) simultaneous
targeted mutagenesis at multiple loci, (2) targeted
chromosomal deletions, (3) synergistic or tunable activation of a gene of interest, (4) synergistic or tunable
Plant Physiol. Vol. 169, 2015

A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation
Figure 1. Applications of the multiplex CRISPR/Cas9 toolbox. The cartoon depicts various scenarios when
applying different components of the
toolbox. A, Simultaneous targeted
mutagenesis at multiple loci in the
same genes or in different genes. B,
Chromosomal deletion. C, Synergistic or tunable transcription activation.
D, Synergistic or tunable transcription
repression. E, Simultaneous activation of multiple genes. F, Simultaneous repression of multiple genes.
repression of a gene of interest, (5) simultaneous activation of multiple genes, and (6) simultaneous repression of multiple genes. We chose to develop a
CRISPR/Cas9 toolbox that allows for all of these applications in both monocot and dicot plants.
T-DNA-based transformation technology is fundamental to modern plant biotechnology, genetics,
molecular biology, and physiology. As such, we developed a method for the assembly of Cas9 (wild type,
nickase, or dCas9) and gRNA(s) into a T-DNA destination vector of interest. The assembly method is
based on both Golden Gate assembly (Engler et al.,
2008) and MultiSite Gateway recombination (Fig. 2A).
Three modules are required for assembly. The rst
module is a Cas9 entry vector, which contains promoterless Cas9 or its derivative genes anked by attL1
and attR5 recombination sites. The second module is a
gRNA entry vector that contains entry gRNA expression cassettes anked by attL5 and attL2 sites. The
third module includes attR1-attR2-containing destination T-DNA vectors that provide promoters of
choice for Cas9 expression. Because such T-DNA
destination vectors have been previously developed

by others (Curtis and Grossniklaus, 2003; Earley et al.,
2006), our work focuses on making entry clones for the
rst two modules (Fig. 2A).
Our Cas9 entry vector module contains eight plasmids (Table I; Supplemental Fig. S1), including three
Cas9 genes that have been previously used in higher
plants. They are plant codon-optimized Cas9 (pcoCas9,
pYPQ150; Li et al., 2013), Arabidopsis codonoptimized Cas9 (AteCas9, pYPQ154; Fauser et al.,
2014; Schiml et al., 2014), human codon-optimized
Cas9 (hSpCas9, pYPQ158; Feng et al., 2013, 2014; Mao
et al., 2013; Zhang et al., 2014), and a plant codonoptimized Cas9 harboring enriched guanine-cytosine
content within key 59 coding regions (Cas9p, pYPQ167;
Ma et al., 2015). Also included within this module is the
D10A nickase version of three out of these four Cas9
genes (pYPQ151, pYPQ155, and pYPQ159), although
testing these nickases is not our focus here. The last
vectors in this module are pYPQ152 and pYPQ153, in
which the deactivated pcoCas9 is fused with the
transcriptional activator VP64 (Beerli et al., 1998) or

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Figure 2. Streamlined assembly of multiplex CRISPR/Cas9 T-DNA vectors. The cartoon illustrates a three-step assembly procedure for construction of different multiplex CRISPR/Cas9 T-DNA vectors for different applications. A, Step 1 involves cloning
individual gRNAs into a series of compatible Golden Gate entry vectors that contain different gRNA promoters. Step 2 involves
assembly of multiple gRNA expression cassettes into one Golden Gate recipient vector, such as pYPQ143 (for three gRNA expression cassettes). Step 3 puts Cas9 clones, gRNA cassettes, and a promoter for Cas9 expression into a single T-DNA binary
vector of choice through Gateway recombination. kan, Kanamycin resistance marker; LacZ, b-galactosidase gene; LB, left border
region; Pol III, RNA polymerase III; RB, right border region; spec, spectinomycin resistance marker; tet, tetracycline resistance
marker. B, A practical timeline for detailed tasks involved in the assembly from the beginning to the end. LR reaction, Invitrogens
LR Clonase II recombination reaction.
three copies of the transcriptional repressor domain

SRDX (Hiratsu et al., 2003), respectively. We examine
these vectors for transcriptional activation and repression of multiple endogenous genes to demonstrate
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unique applications and capabilities of our CRISPR/

Cas9 toolbox.
The gRNA entry vector module contains two sets
of plasmids that enable a two-step Golden Gate

Table I. Vectors in the plant multiplex CRISPR/Cas9 toolbox

Name
Vector Annotation
Addgene No.
pYPQ131A
pYPQ132A
pYPQ133A
pYPQ134A
pYPQ135A
pYPQ136A
pYPQ137A
pYPQ138A
pYPQ131B
pYPQ132B
pYPQ133B
pYPQ131C
pYPQ132C
pYPQ133C
pYPQ131D
pYPQ132D
pYPQ133D
pYPQ141A
pYPQ141B
pYPQ141C
pYPQ141D
pYPQ142
pYPQ143
pYPQ144
pYPQ145
pYPQ146
pYPQ147
pYPQ148
pYPQ150
pYPQ151
pYPQ152
pYPQ153
pYPQ154
pYPQ155
pYPQ158
pYPQ159
pYPQ167
Golden Gate entry vector; first gRNA under AtU6 promoter

Golden Gate entry vector; second gRNA under AtU6 promoter
Golden Gate entry vector; third gRNA under AtU6 promoter
Golden Gate entry vector; fourth gRNA under AtU6 promoter
Golden Gate entry vector; fifth gRNA under AtU6 promoter
Golden Gate entry vector; sixth gRNA under AtU6 promoter
Golden Gate entry vector; seventh gRNA under AtU6 promoter
Golden Gate entry vector; eighth gRNA under AtU6 promoter
Golden Gate entry vector; first gRNA under AtU3 promoter
Golden Gate entry vector; second gRNA under AtU3 promoter
Golden Gate entry vector; third gRNA under AtU3 promoter
Golden Gate entry vector; first gRNA under OsU6 promoter
Golden Gate entry vector; second gRNA under OsU6 promoter
Golden Gate entry vector; third gRNA under OsU6 promoter
Golden Gate entry vector; first gRNA under OsU3 promoter
Golden Gate entry vector; second gRNA under OsU3 promoter
Golden Gate entry vector; third gRNA under OsU3 promoter
Gateway entry vector; single gRNA under AtU6 promoter
Gateway entry vector; single gRNA under AtU3 promoter
Gateway entry vector; single gRNA under OsU6 promoter
Gateway entry vector; single gRNA under OsU3 promoter
Golden Gate recipient and Gateway entry vector; assembly of two gRNAs
Golden Gate recipient and Gateway entry vector; assembly of three gRNAs
Golden Gate recipient and Gateway entry vector; assembly of four gRNAs
Golden Gate recipient and Gateway entry vector; assembly of five gRNAs
Golden Gate recipient and Gateway entry vector; assembly of six gRNAs
Golden Gate recipient and Gateway entry vector; assembly of seven gRNAs
Golden Gate recipient and Gateway entry vector; assembly of eight gRNAs
Gateway entry vector with pcoCas9 (plant codon optimized)
Gateway entry vector with pcoCas9D10A
Gateway entry vector with pco-dCas9-VP64
Gateway entry vector with pco-dCas9-3X(SRDX)
Gateway entry vector with AteCas9 (Arabidopsis codon optimized)
Gateway entry vector with AteCas9D10A
Gateway entry vector with hSpCas9 (human codon optimized)
Gateway entry vector with hSpCas9D10A
Gateway entry vector with Cas9p (plant codon optimized; high guanine-cytosine content at 59 region)
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assembly. This strategy of cloning relies on Type IIS

restriction enzymes that cleave outside their respective recognition sequences (Fig. 2A; Engler et al.,
2008). The rst set of plasmids contain Golden Gate
entry clones, each carrying a complete expression
cassette for one gRNA under either the Arabidopsis
U6 (AtU6) or AtU3 promoter (for dicot plants) or the
rice (Oryza sativa) U6 (OsU6) or OsU3 promoter (for
monocot plants; Fig. 2A; Table I; Fichtner et al., 2014).
The rst step is to clone individual gRNAs into these
Golden Gate entry clones by a simple digestion (with
Type IIS enzyme Esp3I or BsmBI) and ligation step.
This is a single tube reaction and only requires the end
user to input an annealed oligonucleotide pair to
serve as the gRNA molecule of choice (Fig. 2A, Step 1;
for details, see Supplemental Materials and Methods
S1). The second set contains Golden Gate recipient
vectors in which a LacZ gene is readily replaced by
gRNA expression cassettes via Golden Gate reactions
(Fig. 2A; Table I). This is a similar strategy that we
used to assemble TALE repeats (Cermak et al., 2011)
using BsaI as our Type IIS restriction endonuclease

(Fig. 2A, Step 2). gRNA expression cassettes of this
work, however, are much larger (820 bp for AtU6,
720 bp for AtU3, 500 bp for OsU6, and 600 bp for
OsU3) than our previously cloned TAL repeat sequences
(102 bp). Therefore, we needed to ascertain how many
gRNA expression cassettes could be assembled during
a single Golden Gate reaction. To this end, we constructed eight Golden Gate entry vectors harboring
AtU6-based cassettes (pYPQ131ApYPQ138A) and
eight recipient vectors (pYPQ141pYPQ148) for testing Golden Gate assembly for up to eight gRNA cassettes (Table I; Supplemental Fig. S2A). We found
assembly of up to ve gRNA cassettes was readily
achieved (Supplemental Fig. S2B), and the efciency
for assembly of two or three gRNA cassettes was generally over 95% based on blue-white screen. However,
assembly of six or more gRNA cassettes was far less
efcient and often failed (Supplemental Fig. S2B).
We reasoned that expression of three gRNAs should
sufce for many applications. Thus, for AtU3, OsU6,

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Lowder et al.
and OsU3 promoters, we generated Golden Gate

entry clones that allow assembly of up to three
gRNAs (Fig. 1A; Table I). Subsequently, the following
work mainly focuses on testing vectors expressing
three gRNAs.
As illustrated in Figure 2, our assembly of a multiplex CRISPR/Cas9 T-DNA vector takes three steps
and requires very basic molecular biology techniques;
the assembly is readily carried out within 10 d. Importantly, PCR is not used for cloning or validation
throughout the procedure, which reduces the likelihood that mutations will occur within the CRISPR/
Cas9 components. Having established the system,
we next tested our reagents for genome editing and
gene regulation in tobacco, rice (Oryza sativa), and
Arabidopsis.
Targeted Chromosomal Deletions in Tobacco
We rst tested our system for creating targeted

chromosomal deletions in tobacco using an Agrobacterium tumefaciens-mediated transient expression
system in which only a fraction of cells are transformed with our target constructs containing pcoCas9
(Li et al., 2013). Although this system limits our ability
to assay genome-editing efcacy, it allows rapid
testing of multiple assembled gRNAs. FlagellinSensitive2 (FLS3) and Brassinosteroid Kinase1 (BAK1)
are important immune receptor or coreceptor genes
in Arabidopsis (Boller and Felix, 2009; Boller and He,
2009). We identied their orthologs in tobacco,
namely NbFLS2 and NbBAK1. We then designed a
total of 14 gRNAs that target the coding sequences of
both genes for deletions (Supplemental Fig. S3, A
and B). Multiple T-DNA expression vectors were
constructed for the expression of either two or three
gRNAs under expression of the AtU6 or AtU3 promoters. In the case of targeting NbFLS2, expression of two pairs of gRNAs (gR1 and gR2 or gR4
and gR5) both resulted in expected deletions, as
detected by PCR (Supplemental Fig. S3C). Deletions
created by gR1 and gR2 were further conrmed
by DNA sequencing (Supplemental Fig. S3G). Our
data also suggested that the expression of these
two gRNAs was not impacted by having a third
gRNA cassette (NbBAK1-gR3) behind them in the
construct (Supplemental Fig. S3C) or between them
(Supplemental Fig. S3D). We further veried the deletions by sequencing (Supplemental Fig. S3, H and I).
These gRNAs were expressed under an AtU6 promoter. We also tested expression of gRNAs (gR7
and gR9) under an AtU3 promoter (Supplemental
Fig. S3A) and observed these to effectively target
chromosomal deletions (Supplemental Fig. S3, E
and J). Similarly, targeted deletions could be created by expressing multiple gRNAs at a time in
NbBAK1 (Supplemental Fig. S3F), and the deletions
of more than 2 kb were conrmed by sequencing
(Supplemental Fig. S3, K and L). Thus, our multiplex
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CRISPR/Cas9 system allows effective expression of

at least three gRNAs.
Targeted Deletion and Simultaneous Mutagenesis in Rice
Having shown our multiplex Cas9 system works

in dicot species, we next tested the system in a
monocot, namely rice. We aimed to evaluate our
vector system for genome-editing efcacy as well
as to compare the OsU6 and OsU3 promoters. For
this purpose, we generated transgenic protoplasts as
well as stable transgenic plant lines. We chose three
target sites in two genes (Rice Young Seedling Albino
[OsYSA] and Rice Outermost Cell-specic Gene5
[OsROC5]) that have been previously targeted for
mutagenesis (Fig. 3A; Feng et al., 2013). We assembled
two T-DNA constructs that contain the pcoCas9 gene
and expression cassettes for three gRNAs. In both
vectors, each gRNA was arranged in a xed order
(YSA-gR1 at position no. 1, ROC5-gR1 at position no.
2, and YSA-gR2 at position no. 3), with the only difference being their promoters, either OsU6 or OsU3
(Fig. 3B). These two T-DNA constructs were used to
transform rice protoplasts or calli for analysis and
comparison.
We rst tested both T-DNA constructs in a rice
protoplast system. Targeted deletions at the OsYSA
locus were generated using two gRNAs, YSA-gR1 and
YSA-gR2. Both OsU6 and OsU3 promoters effectively yielded targeted deletions of approximately
200 bp, which were clearly detected by PCR (Fig. 3D)
and further conrmed by DNA sequencing (Fig. 3,
F and G). Estimated deletion frequencies in all samples
were as high as approximately 10% (Fig. 4D), indicating comparable efciency for both OsU6 and OsU3.
Next, targeted mutagenesis at all three independent
target sites was determined. Target site anking regions were PCR amplied and followed by restriction
enzyme digestion using SI (for the YSA-gR1 site),
EcoNI (for the YSA-gR2 site), and AhdI (for the ROC5gR1 site). Mutagenesis at these target sites inhibits
PCR product digestion when using these nucleases,
whereas intact, nonmutagenized target sites are
cleaved. Based on this analysis, we found high mutagenesis frequencies at all three sites with either OsU6
or OsU3 promoters (Fig. 3E). Although transformation efciency may not reach 100% in our protoplast
system, the mutation frequencies in harvested protoplasts still ranged from 42% to 67%, suggesting that
multiple target loci could have been simultaneously
modied in any given cell. Furthermore, we validated
mutations at OsROC5 by DNA sequence analysis
(Fig. 3, H and I). After obtaining the high-frequency
genome-editing data in rice protoplasts, we pursued
stable transgenic rice by using the same T-DNA constructs (Fig. 3C). Although the mutagenesis frequency in calli is slightly lower than in protoplasts,
mutants for ysa (albino seedling phenotype; Fig. 3J)
and roc5 (curly leaf phenotype; Fig. 3K) were readily

Figure 3. Targeted deletion and simultaneous mutagenesis in rice. A and B, Illustrations of target genes and relative positions of
gRNA binding sites in OsYSA and OsROC5, respectively. C, An illustration of core components of two T-DNA vectors under study.
Note the fixed positions for three gRNAs with different promoters. NosT, A. tumefaciens nopaline synthase transcriptional terminator sequence. D, PCR-based detection of deletions at OsYSA. The larger bands are the wild type (WT), and the small bands
indicate deletions (marked by asterisks). The deletion frequencies were calculated and listed underneath the gel picture. E, NHEJ
mutations induced by three different gRNAs in OsYSA and OsROC5. The bands representing mutated DNA are indicated by
arrows, while deletion bands at OsYSA are indicated by asterisks. The NHEJ mutagenesis frequencies at all three sites were
calculated and listed underneath the gel picture. F to I, Sequence confirmation of deletions at OsYSA and NHEJ mutations at
OsROC5 with either of the two T-DNA constructs. J, Phenotype of an ysa mutant (albino seedling) that was regenerated from
transformed rice calli harboring the U3-U6-U3 construct. K, Phenotype of a roc5 mutant (curly leaves) that was regenerated from
transformed rice calli harboring the U3-U6-U3 construct. Bars = 1 cm (J) and 2 cm (K).
regenerated from transformed rice calli. By surveying regenerated T0 plants from both constructs
(Fig. 3C), we found the mutation frequencies at individual target sites ranged from 33.3% to 53.3% and
mutations are predominantly biallelic (Supplemental

Table S1). Taken together, the data suggest that our
multiplex CRISPR/Cas9 system is highly active in
monocot rice.

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Lowder et al.
Figure 4. Activation of coding and noncoding

genes by dCas9-VP64 and multiplexed gRNAs. A,
Diagram showing the intron-containing GUS reporter gene is under a synthetic minimal promoter
containing all three target sites from the promoter
of miR319. B, Activation of the GUS reporter gene
by pco-dCas9-VP64 coupled with three gRNAs
that target the synthetic promoter. C and D, These
two diagrams depict gene structure and target sites
of gRNAs for AtPAP1 and miR319, respectively. E
and F, qRT-PCR analysis of target gene transcript
levels. For each experiment, three independent
transgenic lines were randomly chosen for analysis. Transgenic plants that express Cas9 nuclease
with a similar T-DNA vector backbone were used
as negative control (). Error bars represent standard deviations of technical replicates (n = 3).
UTR, Untranslated region.
Targeted Transcriptional Activation of Protein-Coding and

Noncoding Genes by dCas9-VP64 with
Multiplexed gRNAs
A primary design goal for our vector toolbox was to

enable RNA-guided multiplex transcriptional regulation in plants. This capability represents an important
and promising application of the CRISPR/Cas9 system,
and we know of no current toolkits that facilitate this
function in plants. To obtain a Cas9-based transcriptional activator, we fused a VP64 transcriptional
activation domain to the C terminus of deactivated
pcoCas9 (Fig. 1C; Supplemental Fig. S1; Table I). We
reasoned that upon coexpression of this transcriptional
activator and multiple gRNAs targeting a gene promoter of interest, we would be able to increase corresponding gene transcription. To test this strategy in
vivo, we rst generated a reporter T-DNA construct
with an intron-containing GUS gene fused to a minimal
synthetic promoter. The minimal synthetic promoter
contains multiple gRNA binding sites and was
978
designed to test the functionality of our pco-dCas9VP64 transcriptional activator fusion without the
confounding variables of endogenous gene promoter
regulatory elements (Fig. 4A). We then assembled a
T-DNA construct that contains the pco-dCas9-VP64
under the Arabidopsis ubiquitin10 promoter (pUBQ10)
and three gRNAs under the AtU6 promoter (Fig. 4A).
These two T-DNA constructs were used for A. tumefaciensmediated transient expression in tobacco leaves. We
found that coexpression of both constructs resulted in
strong activation of GUS expression, whereas the GUS
reporter construct alone showed little GUS activity
above background (Fig. 4B). This data suggests our
synthetic dCas9-based transcriptional activator functions in a transient expression system.
We next explored transcriptional activation in
Arabidopsis. To determine the efciency of the system
on both protein-coding and non-protein-coding genes,
we chose to target Arabidopsis Production of Anthocyanin
Pigment1 (AtPAP1; encodes a transcription factor;

Borevitz et al., 2000) and miR319 (a microRNA; Weigel

et al., 2000; Palatnik et al., 2003). To promote efcient
transcriptional activation, three gRNAs were designed
to target the promoter of each gene, at sites ranging
from +5 to 306 relative to the transcriptional start site
(Fig. 4, C and D). Two T-DNA constructs were made
with each containing pco-dCas9-VP64 under pUBQ10
control and three gRNAs under the AtU6 promoter.
Transgenic plants were obtained, and three random lines
for each construct were chosen for quantitative real-time
(qRT)-PCR analysis. For AtPAP1, all three lines displayed
transcriptional activation of 2- to 7-fold when compared
with the control plant (Fig. 4E). For miR319, two of three
lines showed gene activation at 3- and 7.5-fold (Fig. 4F).
Collectively, our data demonstrate that Cas9-based
transcriptional activator systems can activate expression
levels of both protein-coding and non-protein-coding
genes in plants. Overexpression of AtPAP1 or miR319
could potentially lead to changes in leaf color (Borevitz
et al., 2000) or leaf morphology (Palatnik et al., 2003).
However, we didnt observe such phenotypes in the lines
we analyzed (Fig. 4), which suggests that higher fold
activation may be required for phenotypic observation. It
is possible to further improve our dCas9-based transcriptional system by recruiting more transcriptional activators, as was recently demonstrated in mammalian
cells (Konermann et al., 2015; Zalatan et al., 2015).
Targeting a Methylated Promoter to Activate an Imprinted
Gene in Arabidopsis
DNA methylation is a prevalent epigenetic modication in plant genomes and commonly methylated
cytosine sites include CpG, CpHpG, and CpHpH (Law

and Jacobsen, 2010). Recent analysis indicates that
about 14% of cytosines are methylated in Arabidopsis
(Capuano et al., 2014). Methylated cytosines in principle should restrict DNA targeting with TALEN and
ZFN. By contrast, such modications should not affect
the Cas9-based DNA targeting system, as recognition is
based on RNA-DNA base pairing. In fact, it was shown
that Cas9-mediated DNA cleavage is unaffected by
DNA methylation in human cells (Hsu et al., 2013).
DNA methylation is a common mechanism used by
plants to turn off transposons and imprinted genes
(Gehring, 2013; Mirouze and Vitte, 2014). We wanted to
test if a Cas9-based transcriptional activator could be
used to reverse methylation-based silencing on plant
gene promoters. It would be a highly valuable tool for
studying and modifying silenced or imprinted genes in
plants.
To investigate if a Cas9-based transcriptional activator can reverse methylation-induced gene silencing,
we targeted an Arabidopsis imprinted gene, AtFIS2
(Luo et al., 2000; Jullien et al., 2006). Arabidopsis
Fertilization-Independent Seed2 (AtFIS2) is silenced in
vegetative tissues, which is likely due to active DNA
methylation within its promoter (Fig. 5A; Jullien et al.,
2006; Hsieh et al., 2009; Zemach et al., 2013). Three
gRNAs were used to target the methylated CpG island
within the AtFIS2 gene promoter. We anticipated that
gRNAs can bind to methylated cytosines and recruit
dCas9-VP64 to activate the transcription of AtFIS2
(Fig. 5B). After analyzing transgenic plants expressing
dCas9-VP64 and target gRNAs, we found signicant
activation of AtFIS2 transcription in Arabidopsis
Figure 5. Activation of a silenced gene by RNA-guided targeting of promoter methylation sites. A, Diagram showing AtFIS2 and its
promoter. A CpG DNA methylation island is indicated using balls and sticks. Partial DNA sequence of the methylation island is
shown. Within the methylation sequence, three gRNA targeting sites are underlined, and CpG methylation sites are marked in red.
AtFIS2 is silenced in rosette leaves due to DNA methylation within the promoter. TSS, Transcription start site. B, Model diagram
showing dCas9-VP64 binding to the DNA methylation island within the promoter and activating AtFIS2 gene expression. Note only
one dCas9-VP64 is depicted. C, Activation of AtFIS2 in rosette leaves of transgenic plants as examined by qRT-PCR analysis. Three
independent transgenic lines were randomly chosen for this analysis. Transgenic plants that express Cas9 nuclease with a similar
T-DNA vector backbone were used as negative controls (). Error bars represent standard deviations of technical replicates (n = 3).

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Lowder et al.
rosette leaves (Fig. 5C). All analyzed lines showed

signicant dCas9-VP64-based gene activation, with
transgenic line number 4 showing about a 400-fold increase in mRNA expression. These data show that our
Cas9-based transcriptional activator system can recognize methylated DNA and signicantly activate silenced genes in plants.
Multiplexed and Simultaneous Gene Repression in
Arabidopsis by dCas9-3X(SRDX)
Our toolbox also enables transcriptional repression

in plants with a synthetic pco-dCas9-3X(SRDX) transcriptional repressor (Fig. 1D; Supplemental Fig. S1;
Table I). We tested this synthetic repressor system on
both protein-coding and noncoding genes in Arabidopsis.
The Arabidopsis Cleavage Stimulating Factor64 (AtCSTF64)
gene encodes an RNA processing factor (Liu et al.,
2010), and we designed three gRNAs targeting its
promoter (Fig. 6A). For testing repression on nonprotein-coding genes, we picked two homologous
and functionally redundant microRNA genes, miR159A
and miR159B (Fig. 6, B and C; Palatnik et al., 2003). We
designed one gRNA targeting miR159A and two gRNAs
targeting mi159B. All three gRNAs were assembled into
a single T-DNA vector harboring pco-dCas9-3X(SRDX)
under pUBQ10 control. Using these constructs, we
evaluated the system for simultaneous multigene
repression.
Both constructs were used to obtain transgenic
plants, and gene expression analysis was carried out
using qRT-PCR. Among three independent transgenic
lines harboring the AtCSTF64 targeting construct, we
detected consistent gene repression, with transcript
levels reduced by approximately 60% compared with
the control (Fig. 6D). Similarly, all three independent

transgenic lines expressing the miR159A/miR159B targeting construct showed signicant reduction of transcript levels for both microRNAs, with transcript levels
being reduced to one-half or even lower (Fig. 6E). Interestingly, repression of miR159A by only one gRNA
worked well when compared with miR159B, which was
targeted by two gRNAs (Fig. 6E). In one line (no. 2), the
repression of miR159A was much stronger than that of
miR159B. We noticed that the gRNA we designed for
miR159A targets a more proximal DNA region relative
to the transcriptional initiation site (Fig. 6B). Target
proximity relative to the transcriptional start site likely
explains the enhanced transcriptional repression we
observed using only a single gRNA targeting miR159A
compared with the two gRNAs targeting miR159B,
which were more distal to the transcriptional start site.
In sum, our data show that multiplex Cas9 systems can
be used for transcriptional repression of both coding
and noncoding genes in plants. Importantly, we show
that multiple genes can be repressed simultaneously by
multiplexed gRNAs.
DISCUSSION
A critical aspect of designing multiplex CRISPR/Cas9

experiments is deciding which strategy to use for expressing multiple gRNAs simultaneously. Different strategies
have been explored, such as a self-processing ribozyme
system (Gao and Zhao, 2014), a transfer RNA-processing
system (Xie et al., 2015), or the Yersinia pestis CRISPR/Cas
Subtype protein4 (Csy4) ribonuclease system (Haurwitz
et al., 2010; Tsai et al., 2014). However, the most popular
approach uses small RNA promoters such as U6 or U3. In
our study, we show the use of small RNA promoters works
Figure 6. Multiplex and simultaneous gene repression in Arabidopsis by dCas9-3X(SRDX). A to

C, These three cartoons depict gene structure and
target sites of gRNAs for AtCSTF64, miR159A,
and miR159B, respectively. D, Targeted repression of AtCSTF64 by pco-dCas9-3X(SRDX) with
coexpression of three multiplex gRNAs. E, Targeted simultaneous repression of two redundant
microRNA genes by pco-dCas9-3X(SRDX) with
coexpression of three gRNAs. For each experiment, three independent transgenic lines were
randomly chosen for analysis. Transgenic plants
expressing only Cas9 nuclease within a similar
T-DNA vector backbone were used as negative
control (). Error bars represent standard deviations of technical replicates (n = 3). UTR, Untranslated region.
980

very well in our system. Although we have focused on

applications with simultaneous expression of multiple
gRNAs, we also include in the kit a set of four vectors
(pYPQ141ApYPQ141D) for expression of only a single gRNA for applications in monocot and dicot plants
(Table I). When using these vectors, one must only follow a simplied procedure, because the Golden Gatebased multivector assembly step (Fig. 2A, Step 2) is no
longer required.
An important issue for targeted mutagenesis using
SSNs is effectively screening for germinal mutations,
which are typically small insertions and deletions.
Generating larger deletions by simultaneous expression
of two gRNA targeting sequences in close proximity
makes such screens more feasible. In fact, simultaneous
targeting of genes using multiple gRNAs has been
demonstrated repeatedly in plants (Li et al., 2013; Mao
et al., 2013; Upadhyay et al., 2013; Brooks et al., 2014;
Gao et al., 2015). In our study, we achieved highfrequency deletions of approximately 200 bp in rice
(Fig. 3D). An alternative approach to achieving relatively large deletions is by using paired Cas9 nickases
(Ran et al., 2013; Ren et al., 2014; Schiml et al., 2014;
Shen et al., 2014). As the functional core of our toolkit
has been validated by experimentation, we opted not to
demonstrate paired nickase activity, but we note that
our toolbox includes Cas9 nickase capability, should
others want to implement this strategy for their own
studies (Table I).
Deletion of very large chromosomal regions containing multiple genes can be a useful tool for researchers,
especially for reducing unwanted or confounding genetic redundancy. Previously, we made large chromosomal deletions in Arabidopsis with ZFNs and
TALENs (Christian et al., 2013; Qi et al., 2013a). To
avoid engineering and simultaneous delivery of multiple ZFNs and TALENs, we used a single pair of ZFNs
or TALENs to target conserved sequences among gene
clusters. This strategy is, however, somewhat cumbersome and has been rendered obsolete by multiplex
CRISPR/Cas9 systems. For example, large chromosomal deletions have recently been demonstrated effectively in rice using CRISPR/Cas9 when two gRNAs
were coexpressed (Zhou et al., 2014). Here, we used
this approach to generate deletions ranging from approximately 300 bp to over 2 kb in tobacco by coexpression
of multiple gRNAs targeting NbFLS2 and NbBAK1
(Supplemental Fig. S3). Thus, our multiplex CRISPR/
Cas9 system is useful for generating large deletions in
plants.
While we were preparing our article, Xing et al.
(2014) and Ma et al. (2015) reported a CRISPR/Cas9
toolkit for targeted mutagenesis in plants. They showed
simultaneous knockout of multiple genes in Arabidopsis
and rice, which, again, demonstrates the versatility and
power of a multiplex CRISPR/Cas9 genome-editing
system. Compared with their toolkits, ours differs signicantly in many aspects. First, our comprehensive
toolbox is designed for both genome editing and transcriptional regulation. To achieve this, we included four
well-characterized Cas9 genes, including the one described by Ma et al. (2015), all of which show high activity in plants (Feng et al., 2013; Li et al., 2013; Fauser
et al., 2014). Aside from codon optimization, these
Cas9 homologs differ from each in epitope tag fusions
(Supplemental Fig. S1). Homologous Cas9 clones differing in codon optimization and epitope tag fusions are
thought to demonstrate variable activity in plant cells
and have different cytotoxic effects in bacteria (Johnson
et al., 2015). Therefore, having multiple Cas9 homologs
to choose from should prove advantageous and
will provide enhanced kit exibility. Second, we made
Cas9D10A nickase versions of three Cas9 clones, which
allow for paired nicking and homologous recombination
while minimizing potential off-target mutagenic effects.
Third, we have included a synthetic transcriptional activator pco-dCas9-VP64 and a synthetic transcriptional
repressor pco-dCas9-3X(SRDX). Fourth, our assembly
strategy positions Cas9 or its variants, gRNA expression
cassettes, and Cas9 promoters into three separate
Gateway-compatible cloning modules. This provides
enhanced exibility to the system, as each module can be
independently changed or upgraded while still maintaining compatibility with other modules. Finally, our
assembly protocol is completely PCR independent,
which ensures high delity and efciency.
In addition to reporting the toolbox and demonstrating its use in plant genome editing, an important
focus in our study is to demonstrate RNA-guided
transcriptional activation and repression in stable
transgenic plants. Very recently, dCas9-based activators and a repressor were tested using transient
expression in tobacco, where different components
were codelivered by multiple T-DNAs (Piatek et al.,
2014). Our work differs signicantly from this work
because we have developed an efcient way to construct all components (promoters, dCas9-based synthetic transcriptional regulators, and multiple gRNA
expression cassettes) into a single T-DNA for easy
delivery (Fig. 2). By focusing on stable transgenic
plants, we showed that both protein-coding and
noncoding genes can be effectively activated (Fig. 4).
Further, we successfully targeted a methylated promoter to activate an imprinted gene (Fig. 5). Our data
thus suggests that CRISPR/Cas9-mediated genome
editing and transcriptional regulation is unaffected
by DNA methylation in plants. Our synthetic repressor also worked at repressing protein-coding or
noncoding genes (Fig. 6). Importantly, we could simultaneously repress two microRNAs (miR159A and
miR159B), even though one microRNA was targeted
by a single gRNA. The scope of gene activation and
repression in our CRISPR/Cas9 system is similar to
those reported in mammalian systems (Gilbert et al.,
2013; Maeder et al., 2013; Perez-Pinera et al., 2013; Qi
et al., 2013a). Taken together, we have shown that all
types of plant endogenous genes are amenable to
transcriptional perturbation using our CRISPR/Cas9
toolbox and the straightforward module assembly
method.

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Lowder et al.
CONCLUSION
In this study, we developed and tested a multifaceted

multiplex CRISPR/Cas9 toolbox that consists of 37
Golden Gate- and Gateway-compatible vectors (Table I;
Supplemental Table S1), which will be made available at
Addgene, Kerafast, and Arabidopsis Biological Resource
Center (ABRC). Based on these vectors, we assembled 29
gRNAs into 14 T-DNA constructs for genome editing
and transcriptional regulation of endogenous genes
in tobacco, rice, and Arabidopsis. To demonstrate multiplexing, we expressed three independent gRNAs simultaneously, an approach that has not yet been explored
much in plants. For, to our knowledge, the rst time, we
successfully activated and repressed transcription of both
protein-coding and noncoding genes and imprinted genes
in Arabidopsis, demonstrating another very important use
of CRISPR/Cas9 in plant research. To this end, we have
successfully demonstrated most applications offered by
this versatile toolbox (Fig. 1). We believe this toolbox will
be very useful in plant basic research and plant synthetic
biology. Its modularity and exibility will allow for easy
and continuous improvement in the future.
MATERIALS AND METHODS
Plant Material and Growth
The wild-type Arabidopsis (Arabidopsis thaliana; ecotype Columbia), tobacco
(Nicotiana benthamiana), and japonica rice (Oryza sativa) Nipponbare were used in
this study. Plants were grown in BM2 soil (Berger) in a growth room or growth
chambers at 25C under a long-day setting (16 h under the light and 8 h in the dark).
Transient Expression in Tobacco

Agrobacterium tumefaciens strain GV3101/pVP90 carrying different expression
T-DNA vectors were cultured at 28C in Luria-Bertani liquid medium supplemented with 50 mg mL1 Gentamycin and 50 mg mL1 Kanamycin. A. tumefaciens
cells were collected by spin at 8,000g for 2 min. The pelleted cells were suspended
with MES buffer (10 mM MES-KOH, 10 mM MgCl2, and 150 mM acetosyringone, pH
5.6) to a nal optical density at 600 nm of 0.2. The bacterial suspension was further
primed by shaking at 150 rpm at 28C for 2 h, and then it was used for inltration of
3- to 4-week-old leaves of tobacco with a 1-mL needleless syringe. The leaf tissue
was collected for DNA extraction or GUS staining 4 d after inltration.
Arabidopsis Transformation and Screen

Arabidopsis ecotype Columbia plants were transformed with T-DNA vectors carried by A. tumefaciens GV3101/pVP90 by following the oral dip protocol (Clough and Bent, 1998). To screen transgenic plants, T1 seeds were
surface sterilized with diluted bleach and kept in 0.1% (w/v) agar in a dark cold
room for 6 d. Then, the seeds were plated onto Murashige and Skoog (MS)
medium, which contains 0.8% (w/v) agar, one-half-strength MS with macroand micronutrients and vitamins (Caisson Labs), 1% (w/v) Suc, 20 mg mL1
Hygromycin B, and 100 mg mL1 Timentin (Gold Biotechnology). One-week-old
transgenic plants were transferred to clean MS plates for growth for another
week before being transplanted into soil.
Rice Protoplast Isolation and Transformation

Rice seeds of japonica Nipponbare were sterilized and germinated in onehalf-strength MS solid medium in a plastic container and grown at 28C in the
dark in a growth chamber for 8 to 10 d. Healthy rice stem and sheath tissues
from 30 to 40 rice seedlings were cut into approximately 0.5- to 1-mm strips. The
strips were transferred into a 90-mm plate and digested in 8 to 10 mL of enzyme
solution (1.5% [w/v] Cellulase R10, 0.75% [w/v] Macerozyme R10, 0.6 M
mannitol, 10 mM MES, pH 5.7, 10 mM CaCl2, and 0.1% [w/v] bovine serum
982
albumin), followed by vacuum inltration for 30 min in the dark using a vacuum
pump at 15 to 20 (in Hg). After a 5- to 6-h digestion with gentle shaking (60
80 rpm), protoplasts were ltered through 40-mm nylon meshes into another
90-mm plate and further transferred into a 50-mL sterile tube. The pellets were
collected by centrifugation at 100g for 5 min and suspended with 10 mL of W5
buffer for washing. Then, the pellets were collected again by centrifugation at 100g
for 2 min and resuspended in Mg-mannitol solution (0.4 M mannitol, 15 mM
MgCl2, and 4 mM MES, pH 5.7). Approximately 5 3 106 cells were used per
transformed experiment. The protoplast transformation was carried out in polyethylene glycol (PEG) solution (40% [w/v] PEG 4000, 0.2 M mannitol, and 0.1 M
CaCl2). The transformation system (30 mg of plasmid DNA mixed with 200 mL of
protoplasts and 230 mL of PEG solution) was gently mixed. After 20-min incubation at room temperature in the dark, 900 mL of W5 buffer was added to stop
transformation. The cells were centrifuged and resuspended in 1 mL of washing/
incubating solution (0.5 M mannitol, 20 mM KCl, and 4 mM MES, pH 5.7) and cultured in six-well plates in the dark at room temperature for 48 h before harvesting.
A. tumefaciens-Mediated Transformation of Rice

Our method is modied from previously published protocols (Nishimura
et al., 2006; Hiei and Komari, 2008). The expression vector (Fig. 3C) was transformed into A. tumefaciens strain EHA105. Mature seeds of japonica rice
Nipponbare were used for stable transformation. First, dehusked seeds were
sterilized with 70% (v/v) ethanol for 1 min and washed ve times with the sterile
water. Then, these seeds were transferred into 2.5% (v/v) sodium hypochlorite,
which contained a drop of Tween 20, to be further sterilized for 15 min. After
being washed ve times, these seeds were sterilized in 2.5% (v/v) sodium hypochlorite again without Tween 20 for 15 min. The seeds were then rinsed ve times
with sterile water. Finally, these seeds were dried on a sterilized lter paper and
cultured on solid medium at 28C under the dark in the growth chamber for 2 to
3 weeks. Actively growing calli were collected for subculture for 1 to 2 weeks at
28C under the dark. Cultured A. tumefaciens cells were collected and resuspended
in liquid medium containing 100 mM acetosyringone (optical density at 600 nm =
0.060.1). Rice calli were immersed in the A. tumefaciens suspension for 30 min.
They were then dried on a sterilized lter paper and cocultured on solid medium
at 25C under the dark in the growth chamber for 3 d. The infected calli were
moved into a sterile plastic bottle and washed ve times with sterile water to removed excessive A. tumefaciens. After being dried on sterilized lter paper, the calli
were transferred onto screening medium at 28C under the dark in the growth
chamber for 5 weeks. During the screening stage, infected calli were transferred
onto fresh screening medium every 2 weeks. After the screening stage, actively
growing calli were moved onto regenerative medium for regeneration at 28C
with a 16-h-light/8-h-dark cycle. After 3 to 4 weeks for regeneration, transgenic seedlings were transferred into sterile plastic bottles containing fresh
solid medium for growing for 2 to 3 weeks before being transferred into soil.
Assays for CRISPR/Cas9 Activity in Vivo

For CRISPR/Cas9-mediated deletions in tobacco, an enrichment PCR procedure was applied. Briey, plant genomic DNA was extracted from transiently
transformed tobacco leaves with the Hexadecyltrimethylammonium bromide (cetyltrimethyl-ammonium bromide) DNA extraction method (Stewart and Via, 1993).
The extracted DNA was digested with EcoRI (for deletions by gR1 and gR2 at
NbFLS2), EcoRI and MfeI (for deletions by gR1 and gR3, by gR4 and gR6, or by gR7
and gR9 at NbFLS2), and BamHI and HindIII (for deletions by gR1 and gR3 or by
gR4 and gR6 at NbBAK1). Then, PCR reactions were conducted using digested
genomic DNA as templates with Taq polymerase (New England Biolabs) and
corresponding primers listed in Supplemental Materials and Methods S1. The
PCR products were resolved in 1.5% (w/v) agarose gel. The bands corresponding
to deletions were excised and cloned into pcr2.1 vector with the TA Cloning Kit
(Life Technologies). Positive clones were picked for sequencing analysis.
For CRISPR/Cas9-mediated deletions and NHEJ mutations in rice, protoplasts transformed with the T-DNA vectors were collected for DNA extraction with the cetyl-trimethyl-ammonium bromide method. The genomic
DNA was used for PCR with KOD FX DNA polymerase (TOYOBO) using
primers YSA-F/R for the OsYSA gene and primers ROC5-F/R for the
OsROC5 gene. OsYSA PCR amplicons were digested with SI or EcoNI, and
OsROC5 PCR amplicons were digested with AhdI. PCR and digested products
were resolved in 2% (w/v) agarose gel. To calculate deletion and NHEJ frequencies, signal intensity of each band from nonsaturated gel pictures were
measured by ImageJ (http://imagej.nih.gov/ij/). The corresponding deletion or
NHEJ mutation bands were also cut and puried with AxyPrep DNA Gel

Extraction Kit (Fisher Scientic). Recovered DNA fragments were cloned into
cloning vector pMD19-T with the pMD19-T Vector Kit (TaKaRa). Positive clones
were picked for sequencing analysis.
GUS Staining
The GUS staining was done by following the procedure described previously
(Baltes et al., 2014).
RNA Extraction and cDNA Synthesis

Fifty to one hundred milligrams of Arabidopsis rosette leaf tissue was collected
from either 2- or 3-week-old hygromycin-resistant seedlings cultured on MS plates
or hygromycin-resistant plants growing on soil. Total RNA was extracted using
TRIzol Reagent (Invitrogen), and homogenization was carried out using a hand
drill and micropestle on dry ice. RNA was isolated and precipitated according
to the TRIzol Reagent product recommendations, with the exception that 1.2 M
NaCl and 0.8 M trisodium citrate (in diethylpyrocarbonate water) and one-half
volume of isopropanol were used to precipitate RNA. Directly following RNA
extraction, contaminating genomic DNA was degraded using DNase I (New
England BioLabs) as recommended by the manufacturer. Complementary DNA
(cDNA) was synthesized from total RNA using the SuperScript III First-Strand
Synthesis System (Invitrogen) and random hexamers.
Quantitative PCR and Data Analysis

Quantitative PCR was carried out using Applied Biosystems SYBR Green
Master Mix (Invitrogen) and cDNA templates (described above) on an Applied
Biosystems 7300 Real-Time PCR System. Disassociation curves of SYBR green
wells were cross checked to eliminate nonspecic, false-positive amplication.
Data are normalized to Actin2 mRNA expression (internal control), and fold
changes are displayed relative to control plant lines using the comparative
threshold cycle method. Error bars represent standard deviations of technical
replicates (n = 3). Refer to Supplemental Materials and Methods S1 for genespecic qRT-PCR primers.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Architecture of Cas9 proteins in the toolbox.
Supplemental Figure S2. Golden Gate assembly of up to 8 gRNA expression cassettes.
Supplemental Figure S3. Targeted chromosomal deletions at NbFLS2 and
NbBAK1 in N. benthamiana.
Supplemental Table S1. Genotyping summary of T0 plants.
Supplemental Materials and Methods S1. Vector assembly protocol and
details.
Supplemental Text S1. Vector DNA sequences and feature annotations in
.gbk format.
ACKNOWLEDGMENTS
We thank Dr. Holger Puchta (Karlsruhe Institute), Dr. Jen Sheen (Harvard
Medical School), Dr. Feng Zhang (Broad Institute of MIT and Harvard), and
Dr. Yao-Guang Liu (South China Agricultural University) for providing Cas9
constructs containing AteCas9, PcoCas9, hSpCas9, and Cas9p, respectively;
and Dr. Baohong Zhang (East Carolina University) and Dr. Xiaoping Pan (East
Carolina University) for providing research equipment and instruments for use.
Received April 29, 2015; accepted August 21, 2015; published August 21, 2015.
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SUPPLEMENTAL MATERIALS AND METHODS
2
3
Protocol for assembly of a multiplex CRISPR/Cas9 T-DNA vector..page 2-3
Plasmids for assembly of different numbers of gRNA expression cassettes.page 4
Sequence and annotation of 37 vectors in the CRISPR/Cas9 toolkit...page 5-44
List of the 15 T-DNA vectors used for plant transformationpage 46
Details on construction of all the vectors in this study....page 47-52
Sequence and annotation of target genes and gRNA sites.page 52-57
List of oligo nucleotides used in this study..page 57-64
10
Synthesized gBlock (IDT) DNA sequences in this studypage 64-66
11
References..page 66
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Protocol for assembly of a multiplex CRISPR/Cas9 T-DNA vector
31
32
33I.
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
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55II.
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75
Step1. Cloning guide RNA (gRNA) into expression vectors

Linearize guide RNA expression plasmids (pYPQ131, 132 and 133; pYPQ141 for single gRNA)
1. First digestion with BglII and SalI (Optional but recommended for zero-background cloning)
H2O
gRNA plasmid (~100 ng/l)
10X NEB buffer 3.1
BglII (10 u/l; NEB)
SalI-HF (10 u/ l; NEB)
Total
14 l
20 l
4 l
1 l
1 l
40 l
37 0C, 3 hrs
2. Second digestion with Esp3I (BsmBI)

Purify 1st digestion products using Qiagen PCR purification kit, elute DNA with 35 l ddH2O, set up
digestion reaction as follow:
Digested gRNA plasmid (from step 1)
32 l
10X OPTIZYME buffer 4
4 l
DTT (20 mM)
2 l
EPS3I (10 u/l; Thermo Scientific)
2 l
Total
40 l
37 0C, O/N
Inactivate enzymes at 800C denature for 20 min, purify the vector using Qiagen PCR purification kit,
and quantify DNA concentration using Nanodrop.
Cloning Oligos into linearized pYPQ131 vector
3. Oligo phosphorylation and annealing
sgRNA oligo forward (100 M)
1 l
sgRNA oligo reverse (100 M)
1 l
10X T4 Polynucleotide Kinase buffer
1 l
T4 Polynucleotide Kinase (10 u/l; NEB)
0.5 l
ddH2O
6.5 l
Total
10 l
Phosphorylate and anneal the oligos using 37 0C for 30 min; 95 0C for 5 min; ramp down to 25 0C at 5 0C
min-1 (i.e., 0.08 0C/second) using a thermocycler (alternatively: cool down in boiled water) .
4. Ligate oligos into linearized gRNA expression vector and transformation of E.coli DH5 cells
ddH2O
6.5 ul
10X NEB T4 ligase buffer
1 l
Linearized gRNA plasmid
1 l
Diluted annealed Oligos (1:200 dilution)
1 l
T4 ligase
0.5 l
Total
10 l
@RT for 1hr
5. Transform E.coli DH5 cells and plate transformed cells on a Tet+ (5ng/ul) LB plate; 37 0C, O/N
2
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
6. Mini-prep two independent clones and verify gRNAs by Sanger sequencing with primer pTC14-F2 (for
pYPQ131, 132 and 133) or M13-F (for pYPQ141).
Step2. Golden Gate Assembly of 2 or 3 guide RNAs

1. Set up Golden Gate reaction as following:
Assembly of 2 guide RNAs
H2O
10X T4 DNA ligase buffer (NEB)
pYPQ142 (100 ng/ l)
pYPQ131-gRNA1 (100 ng/ l)
BsaI (NEB)
T4 DNA ligase (NEB)
Total
Assembly of 3 guide RNAs

H2O
10X T4 DNA ligase buffer (NEB)
pYPQ143 (100 ng/ l)
BsaI (NEB)
T4 DNA ligase (NEB)
Total
2. Run Golden Gate program in a thermocycler as follows:
37 0C, 5 min
10 cycles
16 0C, 10 min
0
50 C, 5 min
80 0C, 5 min
Hold at 10 0C
5 l
1 l
1 l
1 l
1 l
0.5 l
0.5 l
10 l
4 l
1 l
1 l
1 l
1 l
1 l
0.5 l
0.5 l
10 l
3. Transform E. coli DH5 cells and plate transformed cells on a Spe+ (100 g/ml) LB plate.
[Optional: Blue-white screen can be applied because guide RNA expression cassettes will replace
the LacZ gene in pYPQ142 or pYPQ143 recipient plasmid]
4.
Mini-prep two independent clones and verify by restriction digestion
Step 3. Gateway Assembly of Multiplex CRISPR-Cas9 system into a binary vector
106
1. Set up Gateway LR reaction as following:
107
108
109
110
111
112
113
Cas9 entry vector (25ng/ l)

2 l
Guide RNA entry vector (25ng/ l)
2 l
Destination vector (100 ng/ l)
2 l
LR Clonase II
1 l
Total
7 l
@RT for 1hr (O/N recommended)
2. Transform E. coli DH5 cells and plate transformed cells on a Kan+ (50 g/ml) LB plate
3. Mini-prep two independent clones and verify by restriction digestion
114
115
[Note: We have found regular LR Clonase II enzyme is efficient for the Gateway reactions.
There is no need to use MultiSite Gateway recombination kit, which is much more expensive.]
116
117
3
Plasmids for assembly of different numbers of gRNA expression cassettes
118
119
120
Assembly gRNAs for use in dicot plants:

Number of
gRNAs
121
1
2
3
4
5
6
7
8
Gol den
Ga te
cl oni ng
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Gol den Ga te
reci pi ent a nd/or
Ga tewa y entry
pYPQ141A/B
pYPQ142
pYPQ143
pYPQ144
pYPQ145
pYPQ143
pYPQ144
pYPQ145
Gol den Gate entry vectors
pYPQ131A/B
pYPQ131A/B
pYPQ131A/B
pYPQ131A/B
pYPQ131A/B
pYPQ131A/B
pYPQ131A/B
pYPQ132A/B
pYPQ132A/B
pYPQ132A/B
pYPQ132A/B
pYPQ132A/B
pYPQ132A/B
pYPQ132A/B
pYPQ133A/B
pYPQ133A/B
pYPQ133A/B
pYPQ133A/B
pYPQ133A/B
pYPQ133A/B
pYPQ134A
pYPQ134A
pYPQ134A
pYPQ134A
pYPQ134A
pYPQ135A
pYPQ135A
pYPQ135A
pYPQ135A
pYPQ136A
pYPQ136A
pYPQ136A
122
123
Assembly gRNAs for use in monocot plants:

Number
of gRNAs
Golden
Gate
cloning
Golden Gate
recipient and/or
Gateway entry
vector
1
2
3
No
Yes
Yes
pYPQ141C/D
pYPQ142
pYPQ143
Golden Gate entry vectors
pYPQ131C/D
pYPQ131C/D
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
4
pYPQ132C/D
pYPQ132C/D
pYPQ133C/D
pYPQ137A
pYPQ137A
pYPQ138A
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
Sequences of 37 vectors in the CRISPR/Cas9 toolkit

Annotation by color for gRNA expression clones:
BsaI recognition sequence
Overhangs resulted from BsaI cleavage
Promoter for the gRNA
gRNA cloning sites
Terminator for the gRNA
Gateway recombination sites
LacZ promoter and gene
>pYPQ131A (AtU6)
ctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggc
tgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgaattaattctcatgtttg
acagcttatcatcgattagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtc
atcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacagcatcgcca
gtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcctg
ctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggattctctacgccggacgcatcgtggccggcatcacc
ggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtg
ggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttacatgcaccattccttgcggcggcggtgctcaacggcctcaacctacta
ctgggctgcttcctaatgcaggagtcgcataagggagagcgccgacccatgcccttgagagccttcaacccagtcagctccttccggtgggcgcgggg
catgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttc
gctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcgg
cgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatga
ttcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcg
cggctcttaccagcctaacttcgatcattggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtag
gcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacg
gattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttgatcggggaagaacagtatgtcgagctat
tttttgacttactggggatcaagcctgattgggagaaaataaaatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcc
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgctatAGAAATCTCAAAATTCCGG
CAGAACAATTTTGAATCTCGATCCGTAGAAACcAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTT
ACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCT
TGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACA
GCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAcACGAAGTAGTGATTGgagacgagatctagtctgagtc
gaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
GTCGGTGCTTTTTTTGGCAAAAATTTTCAGATTTTTTCTTCATCTGTAGATTTCTGGGTTTTTTTTTCCGTTTCGTGAA
TCATAAGTGAAGTTTTGGATGCAAATCTGCGCGAAAAAAGTTGGACCTGCAATGAGCTTATTTAGATAGCTAAGAC
AAAGTGATTGGTCCGTTGTTTCAGTTCTGATTGTCAGAGAGTTTGTTTCGAGtCGGCGACACCAATGCGTTTTGTTA
ACCAGATTTCGGGTAAGAAATGTATCGAGAGTTTGTTTCcAGACGGCTACATCATTTTCTTATGAAGGGTGAAATT
AGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCcatg
ggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttct
tgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt
tccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcc
tacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag
gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatga
gaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg
gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaa
5
187
188
189
190
191
192
cgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccg
cctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccg
cctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtac
cgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcc
cgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacg
aaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
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211
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213
214
215
216
217
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220
221
222
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226
227
228
229
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232
>pYPQ132A (AtU6)
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgcatgAGAAATCTCAAAATTCCG
GCAGAACAATTTTGAATCTCGATCCGTAGAAACcAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATT
TACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGC
TTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCAC
AGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAcACGAAGTAGTGATTGgagacgagatctagtctgagt
cgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
AGTCGGTGCTTTTTTTGGCAAAAATTTTCAGATTTTTTCTTCATCTGTAGATTTCTGGGTTTTTTTTTCCGTTTCGTGA
ATCATAAGTGAAGTTTTGGATGCAAATCTGCGCGAAAAAAGTTGGACCTGCAATGAGCTTATTTAGATAGCTAAGA
CAAAGTGATTGGTCCGTTGTTTCAGTTCTGATTGTCAGAGAGTTTGTTTCGAGtCGGCGACACCAATGCGTTTTGTT
AACCAGATTTCGGGTAAGAAATGTATCGAGAGTTTGTTTCcAGACGGCTACATCATTTTCTTATGAAGGGTGAAAT
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCgga
cggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc
ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttt
ttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgc
ctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataa
ggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatg
agaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg
ggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaa
acgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattacc
gcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacc
gcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgta
ccgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgc
233
234
ccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaac
gaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
>pYPQ133A (AtU6)
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgggacAGAAATCTCAAAATTCCG
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCcca
gggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc
277
>pYPQ134A (AtU6)
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgccagAGAAATCTCAAAATTCCG
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCtgtt
319
320
321
322
323
324
>pYPQ135A (AtU6)
8
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgtgttAGAAATCTCAAAATTCCGG
CAGAACAATTTTGAATCTCGATCCGTAGAAACcAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTT
ACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCT
TGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACA
GCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAcACGAAGTAGTGATTGgagacgagatctagtctgagtc
GTCGGTGCTTTTTTTGGCAAAAATTTTCAGATTTTTTCTTCATCTGTAGATTTCTGGGTTTTTTTTTCCGTTTCGTGAA
TCATAAGTGAAGTTTTGGATGCAAATCTGCGCGAAAAAAGTTGGACCTGCAATGAGCTTATTTAGATAGCTAAGAC
AAAGTGATTGGTCCGTTGTTTCAGTTCTGATTGTCAGAGAGTTTGTTTCGAGtCGGCGACACCAATGCGTTTTGTTA
ACCAGATTTCGGGTAAGAAATGTATCGAGAGTTTGTTTCcAGACGGCTACATCATTTTCTTATGAAGGGTGAAATT
AGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCtgca
361
362
363
364
365
366
367
368
369
370
371
>pYPQ136A (AtU6)
9
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgtgcaAGAAATCTCAAAATTCCG
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCcgg
tggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
>pYPQ137A (AtU6)
10
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgcggtAGAAATCTCAAAATTCCG
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCgaa
aggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
>pYPQ138A (AtU6)
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcggaaaAGAAATCTCAAAATTCCG
11
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
TAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCtcg
aggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc
487
>pYPQ131B (AtU3)
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgctatTTTACTTTAAATTTTTCTTA
TGGCTCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAcATCTGTTTTGGCTATGTTGG
ACGAAACAAGTGAACTTTTAGGATCAACTTCCGTTTATATACGGAGCTTATATCGAGCAATAAGATAAGTGGGCTT
TTTATGTAATTTAATGGGCTATCGTCCATATATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAG
CTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCACCATTGGTCAg
agacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT
GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTCCCTTTCCTTTTTTCTTTTTTTTGCCATAAACTTAAATTTGTATATC
12
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
GATCATTGTAGATATTGAAAACCTAGAACAAACCAACATCCATGTGAATGTCTTTCATGACTGATTTAGAGATAATT
CTTGAATTTTGGAACTAGAATCTATAATGAGCCTaaattaaaacattgtgcatgggagaccctcgagccacccatgaccaaaatccctt
aacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca
aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatac
caaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct
gctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcac
acagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg
acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcg
ccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttt
tgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga
acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc
tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgctagccaggaagagtttgtagaaacgcaaaaa
ggccatccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaa
atccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttat
ttgatgcctggcagttccctactctcgcgtt
527
>pYPQ132B (AtU3)
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgcatgTTTACTTTAAATTTTTCTTA
CTTGAATTTTGGAACTAGAATCTATAATGAGCCTaaattaaaacattgtgggacggagaccctcgagccacccatgaccaaaatccct
taacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc
aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagata
ccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggc
tgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgca
13
558
559
560
561
562
563
564
565
566
cacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcg
gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttc
gccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcctt
ttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga
567
>pYPQ133B (AtU3)
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgggacTTTACTTTAAATTTTTCTTA
CTTGAATTTTGGAACTAGAATCTATAATGAGCCTaaattaaaacattgtgccagggagaccctcgagccacccatgaccaaaatccctt
caaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct
14
604
605
606
607
608
>pYPQ131C (OsU6-2)
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgctatGGTACCGAGCTCGGATCC
ACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTG
TAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGG
CCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACC
ACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGgagacgagatctagtctgagt
AGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGGTCGAgG
GTATCGATAAGCTTcatgggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgt
agaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccg
gatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccact
tcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactc
aagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgag
atacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagc
gcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg
ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctga
ttctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaa
gagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagc
gcaacgcaattaatacgcgtaccgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctgg
cagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcac
cgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
646
>pYPQ132C (OsU6-2)
15
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgcatgGGTACCGAGCTCGGATCC
GTATCGATAAGCTTggacggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgt
684
>pYPQ133C (OsU6-2)
685
686
687
688
689
690
691
692
16
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgggacGGTACCGAGCTCGGATCC
GTATCGATAAGCTTccagggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgt
722
>pYPQ131D (OsU3)
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
17
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgctatAAGGGATCTTTAAACATAC
GAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTG
CAGTCAGGGACCATAGCACAGGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTG
GGTACGTTGGAAACCACGTGATGTGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGC
CTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCAC
CTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAgagacgagatctagtctgagtc
GTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGGTCGAgGG
TATCGATAAGCTTcatgggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtag
aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgga
tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttc
aagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaa
gacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagat
acctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc
acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg
ggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgatt
ctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag
agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg
caacgcaattaatacgcgtaccgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggc
agtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcacc
gacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
761
>pYPQ132D (OsU3)
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgcatgAAGGGATCTTTAAACATAC
GAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTG
CAGTCAGGGACCATAGCACAGGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTG
GGTACGTTGGAAACCACGTGATGTGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGC
CTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCAC
CTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAgagacgagatctagtctgagtc
18
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
GTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGGTCGAgGG
TATCGATAAGCTTggacggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgta
gaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg
atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccactt
caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactca
agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgaga
tacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcg
cacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg
gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgat
tctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag
caacgcaattaatacgcgtaccgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggc
gacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
800
>pYPQ133D (OsU3)
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
cgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaggtctcgggacAAGGGATCTTTAAACATA
CGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGT
GCAGTCAGGGACCATAGCACAGGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACT
GGGTACGTTGGAAACCACGTGATGTGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAG
CCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCA
CCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAgagacgagatctagtctgag
tcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
GTATCGATAAGCTTccagggagaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgt
19
831
832
833
834
835
836
837
838
839
>pYPQ141A (AtU6)
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
ctagataacgcaggatcctaAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACcAGACGGTC
ATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTT
TAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCC
CATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAG
AcACGAAGTAGTGATTGgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG
CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGGCAAAAATTTTCAGATTTTTTCTTCATC
TGTAGATTTCTGGGTTTTTTTTTCCGTTTCGTGAATCATAAGTGAAGTTTTGGATGCAAATCTGCGCGAAAAAAGTT
GGACCTGCAATGAGCTTATTTAGATAGCTAAGACAAAGTGATTGGTCCGTTGTTTCAGTTCTGATTGTCAGAGAGT
TTGTTTCGAGtCGGCGACACCAATGCGTTTTGTTAACCAGATTTCGGGTAAGAAATGTATCGAGAGTTTGTTTCcAG
ACGGCTACATCATTTTCTTATGAAGGGTGAAATTAGATAGACCAAAGATTGAAACACAACATTTCTTTCACAAAAAT
ATAATAAACTTGATAGCATTTAGGATCAGCaactagtaagggcgaattcgacccagctttcttgtacaaagttggcattataaaaaataa
ttgctcatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgccatccagctgatatcccctatagtgagtcgtattacatgg
tcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctggaccagccag
gacagaaatgcctcgacttcgctgctgcccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgtt
cggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttc
atggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcag
caacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggt
agttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattga
tttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagat
tctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaa
tgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtcc
agcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctg
gcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatg
gagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagtt
ggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacgtgagttac
gcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac
caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcct
tctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggc
gataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagctt
ggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccgg
taagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgactt
gagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgc
tcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca
gcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtt
tcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgctcgccaggaagagtttgtagaaacgcaaaaaggccatccgtcagg
atggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcgg
atttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagtt
20
877
878
ccctactctcgcgttaacgcttgcatggatgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggcccaaataatgattttattttg
actgatagtgacctgttcgttgcaacaaattgatgagcaatgcttttttataatgccaactttgtatacaaaagttgccccatggcgttccct
879
>pYPQ141B (AtU3)
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
ctagataacgcaggatcctaTTTACTTTAAATTTTTCTTATGGCTCAGCCTGTGATGGATAACTGAATCAAACAAATGGCG
TCTGGGTTTAAGAAcATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCCGTTTATATAC
GGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATATATTCACTAATAC
CCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGT
TGTATATATAACACTGAGGGAGCACCATTGGTCAgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAAT
AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTCCCTTTCCTT
TTTTCTTTTTTTTGCCATAAACTTAAATTTGTATATCGATCATTGTAGATATTGAAAACCTAGAACAAACCAACATCC
ATGTGAATGTCTTTCATGACTGATTTAGAGATAATTCTTGAATTTTGGAACTAGAATCTATAATGAGCCTaaattaaaa
cattgtgaactagtaagggcgaattcgacccagctttcttgtacaaagttggcattataaaaaataattgctcatcaatttgttgcaacgaacaggtcac
tatcagtcaaaataaaatcattatttgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtc
tcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctggaccagccaggacagaaatgcctcgacttcgctgctgcccaa
ggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatg
cgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagt
ctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccct
aaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccga
cgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatga
aacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgca
cgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccac
gatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctga
acaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccg
catttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcata
cttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagat
caccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaa
agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaa
gagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag
aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagac
gatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc
tacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacg
agggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggc
ggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgt
ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcg
cccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac
gcaattaatacgcgtaccgctcgccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggcagttt
atggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgaca
aacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgttaacgcttgcatggatgtt
ttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggcccaaataatgattttattttgactgatagtgacctgttcgttgcaacaaattg
atgagcaatgcttttttataatgccaactttgtatacaaaagttgccccatggcgttccct
917
>pYPQ141C (OsU6-2)
918
919
920
921
ctagataacgcaggatccACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTGGATCATGAACCAACGGCCTGGCTG
TATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCG
GGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGG
AACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGgag
21
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
acgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA
AAAAGTGGCACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCG
CTCGAGGTCGAgGGTATCGATAAGCTTgaattcgacccagctttcttgtacaaagttggcattataaaaaataattgctcatcaatttgttg
caacgaacaggtcactatcagtcaaaataaaatcattatttgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctgg
cagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctggaccagccaggacagaaatgcctcg
acttcgctgctgcccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgta
atgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactg
tttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcag
cagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagc
gccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtga
ccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaa
gtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggt
atcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaact
ctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgt
agtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggccc
agtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccact
acgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagc
gtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt
ggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtag
ttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac
cgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgaccta
caccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg
gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt
gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct
gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc
gaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaag
cgggcagtgagcgcaacgcaattaatacgcgtaccgctcgccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgctta
gtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcag
gagagcgttcaccgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgtt
aacgcttgcatggatgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggcccaaataatgattttattttgactgatagtgacct
gttcgttgcaacaaattgatgagcaatgcttttttataatgccaactttgtatacaaaagttgccccatggcgttccct
953
>pYPQ141D (OsU3)
954
955
956
957
958
959
960
961
962
963
964
965
966
967
ctagataacgcaggatcctaAAGGGATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTAT
CAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAGGACAGGCGTCTTCTACTGGT
GCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGGAGTAAGATAAACTGT
AGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCA
ATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGT
GCAGATGATCCGTGGCAgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG
CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATA
TCCATCACACTGGCGGCCGCTCGAGGTCGAgGGTATCGATAAGCTTgaattcgacccagctttcttgtacaaagttggcattataa
aaaataattgctcatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgccatccagctgatatcccctatagtgagtcgta
ttacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctgga
ccagccaggacagaaatgcctcgacttcgctgctgcccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacat
aagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtg
gcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgtta
tggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaacta
22
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
tcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacag
tgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagag
agcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatgg
cagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttg
gtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccga
ctgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgact
gggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgca
gatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacg
tgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca
aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaa
tactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg
ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacaca
gcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggaca
ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcca
cctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgct
ggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacga
ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggc
acgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgctcgccaggaagagtttgtagaaacgcaaaaaggcc
atccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccg
ctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgat
gcctggcagttccctactctcgcgttaacgcttgcatggatgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggcccaaataat
gattttattttgactgatagtgacctgttcgttgcaacaaattgatgagcaatgcttttttataatgccaactttgtatacaaaagttgccccatggcgttc
cct
991
>pYPQ142
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
ctagataacgcaggatccccaagtggtggCTATcgagaccggcgccgctacagggcgcgtcCCATTCGCCATTCAGGCTGCGCAACTG
TTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGAT
TAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACT
CACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTG
CGCGCTTGGCGTAATCATGGTCATagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaag
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctGGACactagtaagggc
gaattcgacccagctttcttgtacaaagttggcattataaaaaataattgctcatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatca
ttatttgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacat
tgcacaagataaaaatatatcatcatgcctcctctggaccagccaggacagaaatgcctcgacttcgctgctgcccaaggttgccgggtgacgcacac
cgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccag
aaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaag
cagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcat
gagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgt
acggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttg
atcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggc
gttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggcta
tcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgc
taaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaa
ccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggctta
tcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaa
taaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttg
agatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttc
23
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
cgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta
catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggc
gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgaga
aagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggga
aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg
ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcc
tttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcct
ctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgc
tcgccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcccgc
caccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaa
ggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgttaacgcttgcatggatgttttcccagtcacgacgttgtaa
aacgacggccagtcttaagctcgggcccaaataatgattttattttgactgatagtgacctgttcgttgcaacaaattgatgagcaatgcttttttataat
gccaactttgtatacaaaagttgccccatggcgttccct
1027
>pYPQ143
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctCCAGactagtaagggc
24
1060
1061
1062
1063
>pYPQ144
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctTGTTactagtaagggc
1099
>pYPQ145
1100
1101
1102
1103
1104
25
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
1131
1132
1133
1134
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctTGCAactagtaagggc
1135
>pYPQ146
1136
1137
1138
1139
1140
1141
1142
1143
1144
1145
1146
1147
1148
1149
1150
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctCGGTactagtaagggc
26
1151
1152
1153
1154
1155
1156
1157
1158
1159
1160
1161
1162
1163
1164
1165
1166
1167
1168
1169
1170
1171
>pYPQ147
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
1189
1190
1191
1192
1193
1194
1195
1196
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctGAAAactagtaagggc
27
1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
1207
>pYPQ148
1208
1209
1210
1211
1212
1213
1214
1215
1216
1217
1218
1219
1220
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
1236
1237
1238
1239
1240
1241
1242
tgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccaccggtggtctctTCGAactagtaagggc
28
1243
1244
1245
1246
1247
1248
1249
1250
Annotation by color for Cas9 expression vectors:
1251
>pYPQ150 (pcoCas9)
1252
1253
1254
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264
1265
1266
1267
1268
1269
1270
1271
1272
1273
1274
1275
1276
1277
1278
1279
1280
1281
1282
1283
1284
1285
1286
1287
1288
gacgtccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaa
gcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaa
atatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattgatcccccctcgacagcttccggaaagggcgaa
ttcgcaactttgtatacaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatac
tgtaaaacacaacatatccagtcactatgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgt
gtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctggaccagccaggacagaaatgcctcgacttcgctgctaccc
aaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgta
tgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtaca
gtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgcc
ctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaacc
gacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgat
gaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtg
cacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagcc
acgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcct
gaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtc
ccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtc
atacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcga
gatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtag
tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttc
caacgcaattaatacgcgtaccgcgagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggc
gacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgttaacgcttgcatgg
atgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggccccaaataatgattttattttgactgatagtgacctgttcgttgcaaca
aattgatgagcaatgcttttttataatgccaactttgtacaaaaaagcaggctccgaattcgcccttcaccATGGATTACAAGGATGATGATG
ATAAGGATTACAAGGATGATGATGATAAGATGGCTCCAAAGAAGAAGAGAAAGGTTGGAATCCACGGAGTTCCA
GCTGCTGATAAGAAGTACTCTATCGGACTTGACATCGGAACCAACTCTGTTGGATGGGCTGTTATCACCGATGAGT
ACAAGGTTCCATCTAAGAAGTTCAAGGTTCTTGGAAACACCGATAGACACTCTATCAAGAAGAACCTTATCGGTGC
TCTTCTTTTCGATTCTGGAGAGACCGCTGAGGCTACCAGATTGAAGAGAACCGCTAGAAGAAGATACACCAGAAG
AAAGAACAGAATCTGCTACCTTCAGGAAATCTTCTCTAACGAGATGGCTAAGGTTGATGATTCTTTCTTCCACAGAC
Gateway recombination sites

Cas9 gene or mutated versions
NOS terminator
VP64 transcription activator
3X (SRDX) transcription repressor
29
1289
1290
1291
1292
1293
1294
1295
1296
1297
1298
1299
1300
1301
1302
1303
1304
1305
1306
1307
1308
1309
1310
1311
1312
1313
1314
1315
1316
1317
1318
1319
1320
1321
1322
1323
1324
1325
1326
1327
1328
1329
1330
1331
1332
1333
1334
1335
1336
TTGAGGAGTCTTTCCTTGTTGAGGAGGATAAGAAGCACGAGAGACACCCAATCTTCGGAAACATCGTTGATGAGG
TTGCTTACCACGAGAAGTACCCAACCATCTACCACCTTAGAAAGAAGTTGGTTGATTCTACCGATAAGGCTGATCTT
AGACTTATCTACCTTGCTCTTGCTCACATGATCAAGTTCAGAGGACACTTCCTTATCGAGGGAGACCTTAACCCAGA
TAACTCTGATGTTGATAAGTTGTTCATCCAGCTTGTTCAGACCTACAACCAGCTTTTCGAGGAGAACCCAATCAACG
CTTCTGGAGTTGATGCTAAGGCTATCCTTTCTGCTAGACTTTCTAAGTCTCGTAGACTTGAGAACCTTATCGCTCAG
CTTCCAGGAGAGAAGAAGAACGGACTTTTCGGAAACCTTATCGCTCTTTCTCTTGGACTTACCCCAAACTTCAAGTC
TAACTTCGATCTTGCTGAGGATGCTAAGTTGCAGCTTTCTAAGGATACCTACGATGATGATCTTGATAACCTTCTTG
CTCAGATCGGAGATCAGTACGCTGATCTTTTCCTTGCTGCTAAGAACCTTTCTGATGCTATCCTTCTTTCTGACATCC
TTAGAGTTAACACCGAGATCACCAAGGCTCCACTTTCTGCTTCTATGATCAAGAGATACGATGAGCACCACCAGGA
TCTTACCCTTTTGAAGGCTCTTGTTAGACAGCAGCTTCCAGAGAAGTACAAGGAAATCTTCTTCGATCAGTCTAAGA
ACGGATACGCTGGATACATCGATGGAGGAGCTTCTCAGGAGGAGTTCTACAAGTTCATCAAGCCAATCCTTGAGA
AGATGGATGGAACCGAGGAGCTTCTTGTTAAGTTGAACAGAGAGGATCTTCTTAGAAAGCAGAGAACCTTCGATA
ACGGATCTATCCCACACCAGATCCACCTTGGAGAGCTTCACGCTATCCTTCGTAGACAGGAGGATTTCTACCCATTC
TTGAAGGATAACAGAGAGAAGATCGAGAAGATCCTTACCTTCAGAATCCCATACTACGTTGGACCACTTGCTAGA
GGAAACTCTCGTTTCGCTTGGATGACCAGAAAGTCTGAGGAGACCATCACCCCTTGGAACTTCGAGGAGgtaagtttc
tgcttctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttcaaaataaaagaatgtagtatatagcaattg
cttttctgtagtttataagtgtgtatattttaatttataacttttctaatatatgaccaaaatttgttgatgtgcagGTTGTTGATAAGGGAGCTTC
TGCTCAGTCTTTCATCGAGAGAATGACCAACTTCGATAAGAACCTTCCAAACGAGAAGGTTCTTCCAAAGCACTCTC
TTCTTTACGAGTACTTCACCGTTTACAACGAGCTTACCAAGGTTAAGTACGTTACCGAGGGAATGAGAAAGCCAGC
TTTCCTTTCTGGAGAGCAGAAGAAGGCTATCGTTGATCTTCTTTTCAAGACCAACAGAAAGGTTACCGTTAAGCAG
TTGAAGGAGGATTACTTCAAGAAGATCGAGTGCTTCGATTCTGTTGAAATCTCTGGAGTTGAGGATAGATTCAACG
CTTCTCTTGGAACCTACCACGATCTTTTGAAGATCATCAAGGATAAGGATTTCCTTGATAACGAGGAGAACGAGGA
CATCCTTGAGGACATCGTTCTTACCCTTACCCTTTTCGAGGATAGAGAGATGATCGAGGAGAGACTCAAGACCTAC
GCTCACCTTTTCGATGATAAGGTTATGAAGCAGTTGAAGAGAAGAAGATACACCGGATGGGGTAGACTTTCTCGT
AAGTTGATCAACGGAATCAGAGATAAGCAGTCTGGAAAGACCATCCTTGATTTCTTGAAGTCTGATGGATTCGCTA
ACAGAAACTTCATGCAGCTTATCCACGATGATTCTCTTACCTTCAAGGAGGACATCCAGAAGGCTCAGGTTTCTGG
ACAGGGAGATTCTCTTCACGAGCACATCGCTAACCTTGCTGGATCTCCAGCTATCAAGAAGGGAATCCTTCAGACC
GTTAAGGTTGTTGATGAGCTTGTTAAGGTTATGGGTAGACACAAGCCAGAGAACATCGTTATCGAGATGGCTAGA
GAGAACCAGACCACCCAGAAGGGACAGAAGAACTCTCGTGAGAGAATGAAGAGAATCGAGGAGGGAATCAAGG
AGCTTGGATCTCAAATCTTGAAGGAGCACCCAGTTGAGAACACCCAGCTTCAGAACGAGAAGTTGTACCTTTACTA
CCTTCAGAACGGAAGAGATATGTACGTTGATCAGGAGCTTGACATCAACAGACTTTCTGATTACGATGTTGATCAC
ATCGTTCCACAGTCTTTCTTGAAGGATGATTCTATCGATAACAAGGTTCTTACCCGTTCTGATAAGAACAGAGGAA
AGTCTGATAACGTTCCATCTGAGGAGGTTGTTAAGAAGATGAAGAACTACTGGAGACAGCTTCTTAACGCTAAGTT
GATCACCCAGAGAAAGTTCGATAACCTTACCAAGGCTGAGAGAGGAGGACTTTCTGAGCTTGATAAGGCTGGATT
CATCAAGAGACAGCTTGTTGAGACCAGACAGATCACCAAGCACGTTGCTCAGATCCTTGATTCTCGTATGAACACC
AAGTACGATGAGAACGATAAGTTGATCAGAGAGGTTAAGGTTATCACCTTGAAGTCTAAGTTGGTTTCTGATTTCA
GAAAGGATTTCCAGTTCTACAAGGTTAGAGAGATCAACAACTACCACCACGCTCACGATGCTTACCTTAACGCTGT
TGTTGGAACCGCTCTTATCAAGAAGTACCCAAAGTTGGAGTCTGAGTTCGTTTACGGAGATTACAAGGTTTACGAT
GTTAGAAAGATGATCGCTAAGTCTGAGCAGGAGATCGGAAAGGCTACCGCTAAGTACTTCTTCTACTCTAACATCA
TGAACTTCTTCAAGACCGAGATCACCCTTGCTAACGGAGAGATCAGAAAGAGACCACTTATCGAGACCAACGGAG
AGACCGGAGAGATCGTTTGGGATAAGGGAAGAGATTTCGCTACCGTTAGAAAGGTTCTTTCTATGCCACAGGTTA
ACATCGTTAAGAAAACCGAGGTTCAGACCGGAGGATTCTCTAAGGAGTCTATCCTTCCAAAGAGAAACTCTGATAA
GTTGATCGCTAGAAAGAAGGATTGGGACCCAAAGAAGTACGGAGGATTCGATTCTCCAACCGTTGCTTACTCTGTT
CTTGTTGTTGCTAAGGTTGAGAAGGGAAAGTCTAAGAAGTTGAAGTCTGTTAAGGAGCTTCTTGGAATCACCATCA
TGGAGCGTTCTTCTTTCGAGAAGAACCCAATCGATTTCCTTGAGGCTAAGGGATACAAGGAGGTTAAGAAGGATC
TTATCATCAAGTTGCCAAAGTACTCTCTTTTCGAGCTTGAGAACGGAAGAAAGAGAATGCTTGCTTCTGCTGGAGA
GCTTCAGAAGGGAAACGAGCTTGCTCTTCCATCTAAGTACGTTAACTTCCTTTACCTTGCTTCTCACTACGAGAAGT
TGAAGGGATCTCCAGAGGATAACGAGCAGAAGCAGCTTTTCGTTGAGCAGCACAAGCACTACCTTGATGAGATCA
30
1337
1338
1339
1340
1341
TCGAGCAAATCTCTGAGTTCTCTAAGAGAGTTATCCTTGCTGATGCTAACCTTGATAAGGTTCTTTCTGCTTACAAC
AAGCACAGAGATAAGCCAATCAGAGAGCAGGCTGAGAACATCATCCACCTTTTCACCCTTACCAACCTTGGTGCTC
CAGCTGCTTTCAAGTACTTCGATACCACCATCGATAGAAAAAGATACACCTCTACCAAGGAGGTTCTTGATGCTACC
CTTATCCACCAGTCTATCACCGGACTTTACGAGACCAGAATCGATCTTTCTCAGCTTGGAGGAGATAAGAGACCAG
CTGCTACCAAGAAGGCTGGACAGGCTAAGAAGAAGAAGTGA
1342
>pYPQ151 (pcoCas9D10A)
1343
1344
1345
1346
1347
1348
1349
1350
1351
1352
1353
1354
1355
1356
1357
1358
1359
1360
1361
1362
1363
1364
1365
1366
1367
1368
1369
1370
1371
1372
1373
1374
1375
1376
1377
1378
1379
1380
1381
1382
GCTGCTGATAAGAAGTACTCTATCGGACTTGCCATCGGAACCAACTCTGTTGGATGGGCTGTTATCACCGATGAGT
31
1383
1384
1385
1386
1387
1388
1389
1390
1391
1392
1393
1394
1395
1396
1397
1398
1399
1400
1401
1402
1403
1404
1405
1406
1407
1408
1409
1410
1411
1412
1413
1414
1415
1416
1417
1418
1419
1420
1421
1422
1423
1424
1425
1426
1427
1428
1429
1430
CCTTCAGAACGGAAGAGATATGTACGTTGATCAGGAGCTTGACATCAACAGACTTTCTGATTACGATGTTGATCAC
32
1431
1432
CTGCTACCAAGAAGGCTGGACAGGCTAAGAAGAAGAAGTGA
1433
>pYPQ152 (pcodCas9-VP64)
1434
1435
1436
1437
1438
1439
1440
1441
1442
1443
1444
1445
1446
1447
1448
1449
1450
1451
1452
1453
1454
1455
1456
1457
1458
1459
1460
1461
1462
1463
1464
1465
1466
1467
1468
1469
1470
1471
1472
1473
1474
1475
1476
33
1477
1478
1479
1480
1481
1482
1483
1484
1485
1486
1487
1488
1489
1490
1491
1492
1493
1494
1495
1496
1497
1498
1499
1500
1501
1502
1503
1504
1505
1506
1507
1508
1509
1510
1511
1512
1513
1514
1515
1516
1517
1518
1519
1520
1521
1522
1523
1524
CCTTCAGAACGGAAGAGATATGTACGTTGATCAGGAGCTTGACATCAACAGACTTTCTGATTACGATGTTGATGCC
CTGCTACCAAGAAGGCTGGACAGGCTAAGAAGAAGAAGGgagacggctctggatcggggtcgggttctggctcaGTCGACGA
TGCTCTTGACGATTTTGACCTCGATATGCTCGACGCTCTTGATGATTTTGATCTCGACATGCTCGATGCACTTGATG
34
1525
1526
1527
1528
1529
1530
1531
ACTTTGACCTTGACATGCTCGACGCACTCGATGACTTCGACCTCGACATGCTTTAGGACGTccgatcgttcaaacatttggc
aataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgca
tgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaat
tatcgcgcgcggtgtcatctatgttactagatcgggaattgatcccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttga
acgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcac
tatgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattg
cacaagataaaaatatatcatcatgcctcct
1532
>pYPQ153 (pcodCas9-3X(SRDX))
1533
1534
1535
1536
1537
1538
1539
1540
1541
1542
1543
1544
1545
1546
1547
1548
1549
1550
1551
1552
1553
1554
1555
1556
1557
1558
1559
1560
1561
1562
1563
1564
1565
1566
1567
1568
1569
1570
35
1571
1572
1573
1574
1575
1576
1577
1578
1579
1580
1581
1582
1583
1584
1585
1586
1587
1588
1589
1590
1591
1592
1593
1594
1595
1596
1597
1598
1599
1600
1601
1602
1603
1604
1605
1606
1607
1608
1609
1610
1611
1612
1613
1614
1615
1616
1617
1618
CCTTCAGAACGGAAGAGATATGTACGTTGATCAGGAGCTTGACATCAACAGACTTTCTGATTACGATGTTGATGCC
36
1619
1620
1621
1622
1623
1624
1625
1626
1627
1628
1629
1630
CTGCTACCAAGAAGGCTGGACAGGCTAAGAAGAAGAAGGGAGACGGCTCTGGATCGGGGTCGGGTTCTGGCTCA
GTCGACCTTGATCTTGACCTCGAACTCAGACTTGGATTTGCTCTCGATCTCGACCTTGAACTTAGACTCGGATTTGC
TCTTGACCTCGATCTTGAGCTTAGACTCGGATTCGCTTAGgacgtccgatcgttcaaacatttggcaataaagtttcttaagattgaat
cctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggttt
ttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgt
tactagatcgggaattgatcccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttgaacgagaaacgtaaaatgatataa
atatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatgccatccagctgatatcccct
atagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatca
tgcctcct
1631
>pYPQ154 (AteCas9)
1632
1633
1634
1635
1636
1637
1638
1639
1640
1641
1642
1643
1644
1645
1646
1647
1648
1649
1650
1651
1652
1653
1654
1655
1656
1657
1658
1659
1660
1661
1662
1663
1664
tggaccagccaggacagaaatgcctcgacttcgctgctacccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtgg
acataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgc
agtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttga
tgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactca
actatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccac
acagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctgg
agagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggaga
atggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttg
ccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgc
ccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgcc
gactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcg
cgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatccctt
caaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct
tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgcgagccaggaagagtttgtagaaacgcaaaaa
ttgatgcctggcagttccctactctcgcgttaacgcttgcatggatgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggcccca
aataatgattttattttgactgatagtgacctgttcgttgcaacaaattgatgagcaatgcttttttataatgccaactttgtacaaaaaagcaggctccg
aattcgcccttcaccATGGATAAGAAGTACTCTATCGGACTCGATATCGGAACTAACTCTGTGGGATGGGCTGTGATCA
CCGATGAGTACAAGGTGCCATCTAAGAAGTTCAAGGTTCTCGGAAACACCGATAGGCACTCTATCAAGAAAAACC
TTATCGGTGCTCTCCTCTTCGATTCTGGTGAAACTGCTGAGGCTACCAGACTCAAGAGAACCGCTAGAAGAAGGTA
CACCAGAAGAAAGAACAGGATCTGCTACCTCCAAGAGATCTTCTCTAACGAGATGGCTAAAGTGGATGATTCATTC
TTCCACAGGCTCGAAGAGTCATTCCTCGTGGAAGAAGATAAGAAGCACGAGAGGCACCCTATCTTCGGAAACATC
GTTGATGAGGTGGCATACCACGAGAAGTACCCTACTATCTACCACCTCAGAAAGAAGCTCGTTGATTCTACTGATA
AGGCTGATCTCAGGCTCATCTACCTCGCTCTCGCTCACATGATCAAGTTCAGAGGACACTTCCTCATCGAGGGTGA
37
1665
1666
1667
1668
1669
1670
1671
1672
1673
1674
1675
1676
1677
1678
1679
1680
1681
1682
1683
1684
1685
1686
1687
1688
1689
1690
1691
1692
1693
1694
1695
1696
1697
1698
1699
1700
1701
1702
1703
1704
1705
1706
1707
1708
1709
1710
1711
1712
TCTCAACCCTGATAACTCTGATGTGGATAAGTTGTTCATCCAGCTCGTGCAGACCTACAACCAGCTTTTCGAAGAGA
ACCCTATCAACGCTTCAGGTGTGGATGCTAAGGCTATCCTCTCTGCTAGGCTCTCTAAGTCAAGAAGGCTTGAGAA
CCTCATTGCTCAGCTCCCTGGTGAGAAGAAGAACGGACTTTTCGGAAACTTGATCGCTCTCTCTCTCGGACTCACCC
CTAACTTCAAGTCTAACTTCGATCTCGCTGAGGATGCAAAGCTCCAGCTCTCAAAGGATACCTACGATGATGATCTC
GATAACCTCCTCGCTCAGATCGGAGATCAGTACGCTGATTTGTTCCTCGCTGCTAAGAACCTCTCTGATGCTATCCT
CCTCAGTGATATCCTCAGAGTGAACACCGAGATCACCAAGGCTCCACTCTCAGCTTCTATGATCAAGAGATACGAT
GAGCACCACCAGGATCTCACACTTCTCAAGGCTCTTGTTAGACAGCAGCTCCCAGAGAAGTACAAAGAGATTTTCT
TCGATCAGTCTAAGAACGGATACGCTGGTTACATCGATGGTGGTGCATCTCAAGAAGAGTTCTACAAGTTCATCAA
GCCTATCCTCGAGAAGATGGATGGAACCGAGGAACTCCTCGTGAAGCTCAATAGAGAGGATCTTCTCAGAAAGCA
GAGGACCTTCGATAACGGATCTATCCCTCATCAGATCCACCTCGGAGAGTTGCACGCTATCCTTAGAAGGCAAGAG
GATTTCTACCCATTCCTCAAGGATAACAGGGAAAAGATTGAGAAGATTCTCACCTTCAGAATCCCTTACTACGTGG
GACCTCTCGCTAGAGGAAACTCAAGATTCGCTTGGATGACCAGAAAGTCTGAGGAAACCATCACCCCTTGGAACTT
CGAAGAGGTGGTGGATAAGGGTGCTAGTGCTCAGTCTTTCATCGAGAGGATGACCAACTTCGATAAGAACCTTCC
AAACGAGAAGGTGCTCCCTAAGCACTCTTTGCTCTACGAGTACTTCACCGTGTACAACGAGTTGACCAAGGTTAAG
TACGTGACCGAGGGAATGAGGAAGCCTGCTTTTTTGTCAGGTGAGCAAAAGAAGGCTATCGTTGATCTCTTGTTCA
AGACCAACAGAAAGGTGACCGTGAAGCAGCTCAAAGAGGATTACTTCAAGAAAATCGAGTGCTTCGATTCAGTTG
AGATTTCTGGTGTTGAGGATAGGTTCAACGCATCTCTCGGAACCTACCACGATCTCCTCAAGATCATTAAGGATAA
GGATTTCTTGGATAACGAGGAAAACGAGGATATCTTGGAGGATATCGTTCTTACCCTCACCCTCTTTGAAGATAGA
GAGATGATTGAAGAAAGGCTCAAGACCTACGCTCATCTCTTCGATGATAAGGTGATGAAGCAGTTGAAGAGAAGA
AGATACACTGGTTGGGGAAGGCTCTCAAGAAAGCTCATTAACGGAATCAGGGATAAGCAGTCTGGAAAGACAAT
CCTTGATTTCCTCAAGTCTGATGGATTCGCTAACAGAAACTTCATGCAGCTCATCCACGATGATTCTCTCACCTTTAA
AGAGGATATCCAGAAGGCTCAGGTTTCAGGACAGGGTGATAGTCTCCATGAGCATATCGCTAACCTCGCTGGATC
TCCTGCAATCAAGAAGGGAATCCTCCAGACTGTGAAGGTTGTGGATGAGTTGGTGAAGGTGATGGGAAGGCATA
AGCCTGAGAACATCGTGATCGAAATGGCTAGAGAGAACCAGACCACTCAGAAGGGACAGAAGAACTCTAGGGAA
AGGATGAAGAGGATCGAGGAAGGTATCAAAGAGCTTGGATCTCAGATCCTCAAAGAGCACCCTGTTGAGAACAC
TCAGCTCCAGAATGAGAAGCTCTACCTCTACTACCTCCAGAACGGAAGGGATATGTATGTGGATCAAGAGTTGGA
TATCAACAGGCTCTCTGATTACGATGTTGATCATATCGTGCCACAGTCATTCTTGAAGGATGATTCTATCGATAACA
AGGTGCTCACCAGGTCTGATAAGAACAGGGGTAAGAGTGATAACGTGCCAAGTGAAGAGGTTGTGAAGAAAATG
AAGAACTATTGGAGGCAGCTCCTCAACGCTAAGCTCATCACTCAGAGAAAGTTCGATAACTTGACTAAGGCTGAG
AGGGGAGGACTCTCTGAATTGGATAAGGCAGGATTCATCAAGAGGCAGCTTGTGGAAACCAGGCAGATCACTAA
GCACGTTGCACAGATCCTCGATTCTAGGATGAACACCAAGTACGATGAGAACGATAAGTTGATCAGGGAAGTGAA
GGTTATCACCCTCAAGTCAAAGCTCGTGTCTGATTTCAGAAAGGATTTCCAATTCTACAAGGTGAGGGAAATCAAC
AACTACCACCACGCTCACGATGCTTACCTTAACGCTGTTGTTGGAACCGCTCTCATCAAGAAGTATCCTAAGCTCGA
GTCAGAGTTCGTGTACGGTGATTACAAGGTGTACGATGTGAGGAAGATGATCGCTAAGTCTGAGCAAGAGATCG
GAAAGGCTACCGCTAAGTATTTCTTCTACTCTAACATCATGAATTTCTTCAAGACCGAGATTACCCTCGCTAACGGT
GAGATCAGAAAGAGGCCACTCATCGAGACAAACGGTGAAACAGGTGAGATCGTGTGGGATAAGGGAAGGGATT
TCGCTACCGTTAGAAAGGTGCTCTCTATGCCACAGGTGAACATCGTTAAGAAAACCGAGGTGCAGACCGGTGGAT
TCTCTAAAGAGTCTATCCTCCCTAAGAGGAACTCTGATAAGCTCATTGCTAGGAAGAAGGATTGGGACCCTAAGAA
ATACGGTGGTTTCGATTCTCCTACCGTGGCTTACTCTGTTCTCGTTGTGGCTAAGGTTGAGAAGGGAAAGAGTAAG
AAGCTCAAGTCTGTTAAGGAACTTCTCGGAATCACTATCATGGAAAGGTCATCTTTCGAGAAGAACCCAATCGATT
TCCTCGAGGCTAAGGGATACAAAGAGGTTAAGAAGGATCTCATCATCAAGCTCCCAAAGTACTCACTCTTCGAACT
CGAGAACGGTAGAAAGAGGATGCTCGCTTCTGCTGGTGAGCTTCAAAAGGGAAACGAGCTTGCTCTCCCATCTAA
GTACGTTAACTTTCTTTACCTCGCTTCTCACTACGAGAAGTTGAAGGGATCTCCAGAAGATAACGAGCAGAAGCAA
CTTTTCGTTGAGCAGCACAAGCACTACTTGGATGAGATCATCGAGCAGATCTCTGAGTTCTCTAAAAGGGTGATCC
TCGCTGATGCAAACCTCGATAAGGTGTTGTCTGCTTACAACAAGCACAGAGATAAGCCTATCAGGGAACAGGCAG
AGAACATCATCCATCTCTTCACCCTTACCAACCTCGGTGCTCCTGCTGCTTTCAAGTACTTCGATACAACCATCGATA
GGAAGAGATACACCTCTACCAAAGAAGTGCTCGATGCTACCCTCATCCATCAGTCTATCACTGGACTCTACGAGAC
TAGGATCGATCTCTCACAGCTCGGTGGTGATTCAAGGGCTGATCCTAAGAAGAAGAGGAAGGTTTGAggcgcgccga
38
1713
1714
1715
1716
1717
1718
gctctctagagctagcgtttaaacaccggtgacgtcccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatg
attatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaa
ttatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattgatc
ccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattag
attttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatgccatccagctgatatcccctatagtgagtcgtattacatg
gtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctc
1719
>pYPQ155 (AteCas9D10A)
1720
1721
1722
1723
1724
1725
1726
1727
1728
1729
1730
1731
1732
1733
1734
1735
1736
1737
1738
1739
1740
1741
1742
1743
1744
1745
1746
1747
1748
1749
1750
1751
1752
1753
1754
1755
1756
1757
1758
aattcgcccttcaccATGGATAAGAAGTACTCTATCGGACTCGCTATCGGAACTAACTCTGTGGGATGGGCTGTGATCA
CCGATGAGTACAAGGTGCCATCTAAGAAGTTCAAGGTTCTCGGAAACACCGATAGGCACTCTATCAAGAAAAACC
TTATCGGTGCTCTCCTCTTCGATTCTGGTGAAACTGCTGAGGCTACCAGACTCAAGAGAACCGCTAGAAGAAGGTA
CACCAGAAGAAAGAACAGGATCTGCTACCTCCAAGAGATCTTCTCTAACGAGATGGCTAAAGTGGATGATTCATTC
TTCCACAGGCTCGAAGAGTCATTCCTCGTGGAAGAAGATAAGAAGCACGAGAGGCACCCTATCTTCGGAAACATC
GTTGATGAGGTGGCATACCACGAGAAGTACCCTACTATCTACCACCTCAGAAAGAAGCTCGTTGATTCTACTGATA
AGGCTGATCTCAGGCTCATCTACCTCGCTCTCGCTCACATGATCAAGTTCAGAGGACACTTCCTCATCGAGGGTGA
TCTCAACCCTGATAACTCTGATGTGGATAAGTTGTTCATCCAGCTCGTGCAGACCTACAACCAGCTTTTCGAAGAGA
ACCCTATCAACGCTTCAGGTGTGGATGCTAAGGCTATCCTCTCTGCTAGGCTCTCTAAGTCAAGAAGGCTTGAGAA
CCTCATTGCTCAGCTCCCTGGTGAGAAGAAGAACGGACTTTTCGGAAACTTGATCGCTCTCTCTCTCGGACTCACCC
CTAACTTCAAGTCTAACTTCGATCTCGCTGAGGATGCAAAGCTCCAGCTCTCAAAGGATACCTACGATGATGATCTC
GATAACCTCCTCGCTCAGATCGGAGATCAGTACGCTGATTTGTTCCTCGCTGCTAAGAACCTCTCTGATGCTATCCT
CCTCAGTGATATCCTCAGAGTGAACACCGAGATCACCAAGGCTCCACTCTCAGCTTCTATGATCAAGAGATACGAT
39
1759
1760
1761
1762
1763
1764
1765
1766
1767
1768
1769
1770
1771
1772
1773
1774
1775
1776
1777
1778
1779
1780
1781
1782
1783
1784
1785
1786
1787
1788
1789
1790
1791
1792
1793
1794
1795
1796
1797
1798
1799
1800
1801
1802
1803
1804
1805
1806
GAGCACCACCAGGATCTCACACTTCTCAAGGCTCTTGTTAGACAGCAGCTCCCAGAGAAGTACAAAGAGATTTTCT
TCGATCAGTCTAAGAACGGATACGCTGGTTACATCGATGGTGGTGCATCTCAAGAAGAGTTCTACAAGTTCATCAA
GCCTATCCTCGAGAAGATGGATGGAACCGAGGAACTCCTCGTGAAGCTCAATAGAGAGGATCTTCTCAGAAAGCA
GAGGACCTTCGATAACGGATCTATCCCTCATCAGATCCACCTCGGAGAGTTGCACGCTATCCTTAGAAGGCAAGAG
GATTTCTACCCATTCCTCAAGGATAACAGGGAAAAGATTGAGAAGATTCTCACCTTCAGAATCCCTTACTACGTGG
GACCTCTCGCTAGAGGAAACTCAAGATTCGCTTGGATGACCAGAAAGTCTGAGGAAACCATCACCCCTTGGAACTT
CGAAGAGGTGGTGGATAAGGGTGCTAGTGCTCAGTCTTTCATCGAGAGGATGACCAACTTCGATAAGAACCTTCC
AAACGAGAAGGTGCTCCCTAAGCACTCTTTGCTCTACGAGTACTTCACCGTGTACAACGAGTTGACCAAGGTTAAG
TACGTGACCGAGGGAATGAGGAAGCCTGCTTTTTTGTCAGGTGAGCAAAAGAAGGCTATCGTTGATCTCTTGTTCA
AGACCAACAGAAAGGTGACCGTGAAGCAGCTCAAAGAGGATTACTTCAAGAAAATCGAGTGCTTCGATTCAGTTG
AGATTTCTGGTGTTGAGGATAGGTTCAACGCATCTCTCGGAACCTACCACGATCTCCTCAAGATCATTAAGGATAA
GGATTTCTTGGATAACGAGGAAAACGAGGATATCTTGGAGGATATCGTTCTTACCCTCACCCTCTTTGAAGATAGA
GAGATGATTGAAGAAAGGCTCAAGACCTACGCTCATCTCTTCGATGATAAGGTGATGAAGCAGTTGAAGAGAAGA
AGATACACTGGTTGGGGAAGGCTCTCAAGAAAGCTCATTAACGGAATCAGGGATAAGCAGTCTGGAAAGACAAT
CCTTGATTTCCTCAAGTCTGATGGATTCGCTAACAGAAACTTCATGCAGCTCATCCACGATGATTCTCTCACCTTTAA
AGAGGATATCCAGAAGGCTCAGGTTTCAGGACAGGGTGATAGTCTCCATGAGCATATCGCTAACCTCGCTGGATC
TCCTGCAATCAAGAAGGGAATCCTCCAGACTGTGAAGGTTGTGGATGAGTTGGTGAAGGTGATGGGAAGGCATA
AGCCTGAGAACATCGTGATCGAAATGGCTAGAGAGAACCAGACCACTCAGAAGGGACAGAAGAACTCTAGGGAA
AGGATGAAGAGGATCGAGGAAGGTATCAAAGAGCTTGGATCTCAGATCCTCAAAGAGCACCCTGTTGAGAACAC
TCAGCTCCAGAATGAGAAGCTCTACCTCTACTACCTCCAGAACGGAAGGGATATGTATGTGGATCAAGAGTTGGA
TATCAACAGGCTCTCTGATTACGATGTTGATCATATCGTGCCACAGTCATTCTTGAAGGATGATTCTATCGATAACA
AGGTGCTCACCAGGTCTGATAAGAACAGGGGTAAGAGTGATAACGTGCCAAGTGAAGAGGTTGTGAAGAAAATG
AAGAACTATTGGAGGCAGCTCCTCAACGCTAAGCTCATCACTCAGAGAAAGTTCGATAACTTGACTAAGGCTGAG
AGGGGAGGACTCTCTGAATTGGATAAGGCAGGATTCATCAAGAGGCAGCTTGTGGAAACCAGGCAGATCACTAA
GCACGTTGCACAGATCCTCGATTCTAGGATGAACACCAAGTACGATGAGAACGATAAGTTGATCAGGGAAGTGAA
GGTTATCACCCTCAAGTCAAAGCTCGTGTCTGATTTCAGAAAGGATTTCCAATTCTACAAGGTGAGGGAAATCAAC
AACTACCACCACGCTCACGATGCTTACCTTAACGCTGTTGTTGGAACCGCTCTCATCAAGAAGTATCCTAAGCTCGA
GTCAGAGTTCGTGTACGGTGATTACAAGGTGTACGATGTGAGGAAGATGATCGCTAAGTCTGAGCAAGAGATCG
GAAAGGCTACCGCTAAGTATTTCTTCTACTCTAACATCATGAATTTCTTCAAGACCGAGATTACCCTCGCTAACGGT
GAGATCAGAAAGAGGCCACTCATCGAGACAAACGGTGAAACAGGTGAGATCGTGTGGGATAAGGGAAGGGATT
TCGCTACCGTTAGAAAGGTGCTCTCTATGCCACAGGTGAACATCGTTAAGAAAACCGAGGTGCAGACCGGTGGAT
TCTCTAAAGAGTCTATCCTCCCTAAGAGGAACTCTGATAAGCTCATTGCTAGGAAGAAGGATTGGGACCCTAAGAA
ATACGGTGGTTTCGATTCTCCTACCGTGGCTTACTCTGTTCTCGTTGTGGCTAAGGTTGAGAAGGGAAAGAGTAAG
AAGCTCAAGTCTGTTAAGGAACTTCTCGGAATCACTATCATGGAAAGGTCATCTTTCGAGAAGAACCCAATCGATT
TCCTCGAGGCTAAGGGATACAAAGAGGTTAAGAAGGATCTCATCATCAAGCTCCCAAAGTACTCACTCTTCGAACT
CGAGAACGGTAGAAAGAGGATGCTCGCTTCTGCTGGTGAGCTTCAAAAGGGAAACGAGCTTGCTCTCCCATCTAA
GTACGTTAACTTTCTTTACCTCGCTTCTCACTACGAGAAGTTGAAGGGATCTCCAGAAGATAACGAGCAGAAGCAA
CTTTTCGTTGAGCAGCACAAGCACTACTTGGATGAGATCATCGAGCAGATCTCTGAGTTCTCTAAAAGGGTGATCC
TCGCTGATGCAAACCTCGATAAGGTGTTGTCTGCTTACAACAAGCACAGAGATAAGCCTATCAGGGAACAGGCAG
AGAACATCATCCATCTCTTCACCCTTACCAACCTCGGTGCTCCTGCTGCTTTCAAGTACTTCGATACAACCATCGATA
GGAAGAGATACACCTCTACCAAAGAAGTGCTCGATGCTACCCTCATCCATCAGTCTATCACTGGACTCTACGAGAC
TAGGATCGATCTCTCACAGCTCGGTGGTGATTCAAGGGCTGATCCTAAGAAGAAGAGGAAGGTTTGAggcgcgccga
gctctctagagctagcgtttaaacaccggtgacgtcccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatg
attatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaa
ttatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattgatc
ccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattag
attttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatgccatccagctgatatcccctatagtgagtcgtattacatg
gtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctc
40
1807
>pYPQ158 (hSpCas9)
1808
1809
1810
1811
1812
1813
1814
1815
1816
1817
1818
1819
1820
1821
1822
1823
1824
1825
1826
1827
1828
1829
1830
1831
1832
1833
1834
1835
1836
1837
1838
1839
1840
1841
1842
1843
1844
1845
1846
1847
1848
1849
1850
1851
1852
1853
aattcgcccttcaccatgAccggtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACA
AAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA
GAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGC
CCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGT
TCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA
CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGA
AGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGG
CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGC
GGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGA
CAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAA
CGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCG
CCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACT
TCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGAC
AACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGC
TGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACG
AGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCT
TCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA
AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAG
CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG
GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACG
41
1854
1855
1856
1857
1858
1859
1860
1861
1862
1863
1864
1865
1866
1867
1868
1869
1870
1871
1872
1873
1874
1875
1876
1877
1878
1879
1880
1881
1882
1883
1884
1885
1886
1887
1888
1889
1890
1891
1892
1893
1894
1895
1896
1897
TGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGG
AACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC
CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAA
GTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCT
GCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGA
CTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC
AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTT
GAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCT
GAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCC
GGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACA
GCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCA
ATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTG
ATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA
AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACA
CCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGT
GGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGA
CGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG
AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGAC
AATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGA
AACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACA
AGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTA
CAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGAT
CAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCG
CCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGAC
CGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCG
TGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAG
ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAG
AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGC
CAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA
AGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATC
AAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG
AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAG
GGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAG
CAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG
CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCT
GCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACC
CTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCG
GCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTGTGATAAacgtcccgatcgttcaaacatttggcaata
aagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgac
gttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatc
gcgcgcggtgtcatctatgttactagatcgggaattgatcccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttgaacga
gaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatg
ccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcaca
agataaaaatatatcatcatgcctcctc
1898
>pYPQ159 (hSpCas9D10A)
42
1899
1900
1901
1902
1903
1904
1905
1906
1907
1908
1909
1910
1911
1912
1913
1914
1915
1916
1917
1918
1919
1920
1921
1922
1923
1924
1925
1926
1927
1928
1929
1930
1931
1932
1933
1934
1935
1936
1937
1938
1939
1940
1941
1942
1943
1944
1945
1946
aattcgcccttcaccatgAccggtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACA
AAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA
GAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCC
CAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTT
CGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAAC
CGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAA
GAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC
CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCG
GCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGAC
AACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAAC
GCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGC
CCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTT
CAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACA
ACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCT
GAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGA
GCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTT
CGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA
AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAG
CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG
GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACG
TGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGG
AACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC
43
1947
1948
1949
1950
1951
1952
1953
1954
1955
1956
1957
1958
1959
1960
1961
1962
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAA
GTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCT
GCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGA
CTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC
AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTT
GAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCT
GAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCC
GGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACA
GCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCA
ATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTG
ATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA
AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACA
CCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGT
GGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGA
CGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG
AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGAC
AATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGA
AACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACA
AGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTA
CAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGAT
CAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCG
CCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGAC
CGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCG
TGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAG
ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAG
AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGC
CAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGA
AGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATC
AAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG
AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAG
GGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAG
CAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG
CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCT
GCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACC
CTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCG
GCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGAATTGTGATAAacgtcccgatcgttcaaacatttggcaata
aagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgac
gttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatc
gcgcgcggtgtcatctatgttactagatcgggaattgatcccccctcgacagcttccggaaagggcgaattcgcaactttgtatacaaaagttgaacga
gaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatg
ccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcaca
agataaaaatatatcatcatgcctcctc
1989
1990
>pYPQ167 (Cas9p)
1991
1992
44
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
2025
2026
2027
2028
2029
2030
2031
2032
2033
2034
2035
2036
2037
2038
2039
2040
aattcgcccttcaccatggctcctaagaagaagcggaaggttggtattcacggggtgcctgcggctgacaagaagtactccatcggcctcgacatcgg
caccaacagcgtcggctgggcggtgatcaccgacgagtacaaggtcccgtccaagaagttcaaggtcctgggcaacaccgaccgccactccatcaag
aagaacctcatcggcgccctcctcttcgactccggcgagacggcggaggcgacccgcctcaagcgcaccgcccgccgccgctacacccgccgcaaga
accgcatctgctacctccaggagatcttctccaacgagatggcgaaggtcgacgactccttcttccaccgcctcgaggagtccttcctcgtggaggagg
acaagaagcacgagcgccaccccatcttcggcaacatcgtcgacgaggtcgcctaccacgagaagtaccccactatctaccaccttcgtaagaagctt
gttgactctactgataaggctgatcttcgtctcatctaccttgctctcgctcacatgatcaagttccgtggtcacttccttatcgagggtgaccttaaccct
gataactccgacgtggacaagctcttcatccagctcgtccagacctacaaccagctcttcgaggagaaccctatcaacgcttccggtgtcgacgctaag
gcgatcctttccgctaggctctccaagtccaggcgtctcgagaacctcatcgcccagctccctggtgagaagaagaacggtcttttcggtaacctcatcg
ctctctccctcggtctgacccctaacttcaagtccaacttcgacctcgctgaggacgctaagcttcagctctccaaggatacctacgacgatgatctcga
caacctcctcgctcagattggagatcagtacgctgatctcttccttgctgctaagaacctctccgatgctatcctcctttcggatatccttagggttaacac
tgagatcactaaggctcctctttctgcttccatgatcaagcgctacgacgagcaccaccaggacctcaccctcctcaaggctcttgttcgtcagcagctc
cccgagaagtacaaggagatcttcttcgaccagtccaagaacggctacgccggttacattgacggtggagctagccaggaggagttctacaagttcat
caagccaatccttgagaagatggatggtactgaggagcttctcgttaagcttaaccgtgaggacctccttaggaagcagaggactttcgataacggct
ctatccctcaccagatccaccttggtgagcttcacgccatccttcgtaggcaggaggacttctaccctttcctcaaggacaaccgtgagaagatcgaga
agatccttactttccgtattccttactacgttggtcctcttgctcgtggtaactcccgtttcgcttggatgactaggaagtccgaggagactatcacccctt
ggaacttcgaggaggttgttgacaagggtgcttccgcccagtccttcatcgagcgcatgaccaacttcgacaagaacctccccaacgagaaggtcctc
cccaagcactccctcctctacgagtacttcacggtctacaacgagctcaccaaggtcaagtacgtcaccgagggtatgcgcaagcctgccttcctctcc
ggcgagcagaagaaggctatcgttgacctcctcttcaagaccaaccgcaaggtcaccgtcaagcagctcaaggaggactacttcaagaagatcgagt
gcttcgactccgtcgagatcagcggcgttgaggaccgtttcaacgcttctctcggtacctaccacgatctcctcaagatcatcaaggacaaggacttcct
cgacaacgaggagaacgaggacatcctcgaggacatcgtcctcactcttactctcttcgaggatagggagatgatcgaggagaggctcaagacttac
gctcatctcttcgatgacaaggttatgaagcagctcaagcgtcgccgttacaccggttggggtaggctctcccgcaagctcatcaacggtatcagggat
aagcagagcggcaagactatcctcgacttcctcaagtctgatggtttcgctaacaggaacttcatgcagctcatccacgatgactctcttaccttcaagg
aggatattcagaaggctcaggtgtccggtcagggcgactctctccacgagcacattgctaaccttgctggttcccctgctatcaagaagggcatccttc
agactgttaaggttgtcgatgagcttgtcaaggttatgggtcgtcacaagcctgagaacatcgtcatcgagatggctcgtgagaaccagactacccag
45
2041
2042
2043
2044
2045
2046
2047
2048
2049
2050
2051
2052
2053
2054
2055
2056
2057
2058
2059
2060
2061
2062
2063
2064
2065
aagggtcagaagaactcgagggagcgcatgaagaggattgaggagggtatcaaggagcttggttctcagatccttaaggagcaccctgtcgagaac
acccagctccagaacgagaagctctacctctactacctccagaacggtagggatatgtacgttgaccaggagctcgacatcaacaggctttctgacta
cgacgtcgaccacattgttcctcagtctttccttaaggatgactccatcgacaacaaggtcctcacgaggtccgacaagaacaggggtaagtcggaca
acgtcccttccgaggaggttgtcaagaagatgaagaactactggaggcagcttctcaacgctaagctcattacccagaggaagttcgacaacctcac
gaaggctgagaggggtggcctttccgagcttgacaaggctggtttcatcaagaggcagcttgttgagacgaggcagattaccaagcacgttgctcaga
tcctcgattctaggatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtcaaggtgatcaccctcaagtccaagctcgtctccgacttc
cgcaaggacttccagttctacaaggtccgcgagatcaacaactaccaccacgctcacgatgcttaccttaacgctgtcgttggtaccgctcttatcaag
aagtaccctaagcttgagtccgagttcgtctacggtgactacaaggtctacgacgttcgtaagatgatcgccaagtccgagcaggagatcggcaaggc
caccgccaagtacttcttctactccaacatcatgaacttcttcaagaccgagatcaccctcgccaacggcgagatccgcaagcgccctcttatcgagac
gaacggtgagactggtgagatcgtttgggacaagggtcgcgacttcgctactgttcgcaaggtcctttctatgcctcaggttaacatcgtcaagaagac
cgaggtccagaccggtggcttctccaaggagtctatccttccaaagagaaactcggacaagctcatcgctaggaagaaggattgggaccctaagaag
tacggtggtttcgactcccctactgtcgcctactccgtcctcgtggtcgccaaggtggagaagggtaagtcgaagaagctcaagtccgtcaaggagctc
ctcggcatcaccatcatggagcgctcctccttcgagaagaacccgatcgacttcctcgaggccaagggctacaaggaggtcaagaaggacctcatca
tcaagctccccaagtactctcttttcgagctcgagaacggtcgtaagaggatgctggcttccgctggtgagctccagaagggtaacgagcttgctcttcc
ttccaagtacgtgaacttcctctacctcgcctcccactacgagaagctcaagggttcccctgaggataacgagcagaagcagctcttcgtggagcagc
acaagcactacctcgacgagatcatcgagcagatctccgagttctccaagcgcgtcatcctcgctgacgctaacctcgacaaggtcctctccgcctaca
acaagcaccgcgacaagcccatccgcgagcaggccgagaacatcatccacctcttcacgctcacgaacctcggcgcccctgctgctttcaagtacttc
gacaccaccatcgacaggaagcgttacacgtccaccaaggaggttctcgacgctactctcatccaccagtccatcaccggtctttacgagactcgtatc
gacctttcccagcttggtggtgataagcgtcctgctgccaccaaaaaggccggacaggctaagaaaaagaagtaggatccacctgatctagagctag
cgtttaaacaccggtgacgtcccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttc
tgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacg
cgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattgatcccccctcgacagctt
ccggaaagggcgaattcgcaactttgtatacaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaa
cagactacataatactgtaaaacacaacatatccagtcactatgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcct
ggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctc
2066
2067
List of the 15 T-DNA vectors used for plant transformation:

Name of T DNA construct
pMDC32-pcoCas9-NbFLS2-gR1-gR2
pMDC32-pcoCas9-NbFLS2-gR1-gR2-NbBAK1-gR3
pMDC32-pcoCas9-NbFLS2-gR1-gR2-gR3
pMDC32-pcoCas9-NbFLS2-gR4-gR5-gR6
pMDC32-pcoCas9-NbFLS2-gR7-gR9
pMDC32-pcoCas9-NbBAK1-gR1-gR2-gR3
pMDC32-pcoCas9-NbBAK1-gR4-gR5-gR6
pMDC32-pcoCas9-OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U6U3U6)
pMDC32-pcoCas9-OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U3U6U3)
pMDC99-SynPro-GUS-intron
pYPQ202-pcodCas9-VP64-AtPAP1-gR1-gR2-gR3
pYPQ202-pcodCas9-VP64-miR319-gR1-gR2-gR3
pYPQ202-pcodCas9-VP64-AtFIS2-gR1-gR2-gR3
pYPQ202-pcodCas9-3X(SRDX)-AtCSTF64-gR1-gR2-gR3
pYPQ202-pcodCas9-3X(SRDX)-miR159A-gR2-miR159B-gR1-gR2
46
Corresponding data
Figure S3
Figure S3
Figure S3
Figure S3
Figure S3
Figure S3
Figure S3
Figure 3
Figure 3
Figure 4
Figure 4
Figure 4
Figure 5
Figure 6
Figure 6
2068
Details on construction of the vectors in this study
2069
2070
2071
2072
Note: All the restriction enzymes, T4 ligase, T4 Polynucleotide kinase, and Gibson Assembly cloning kit
were purchased from NEB. Esp3I (BsmBI) is from Thermo Scientific. The miniprep kit, gel extraction kit
and PCR purification kit are all from Qiagen. Synthetic DNA elements and genes (gBlocks) are from
Integrated DNA Technologies (IDT).
2073
A.
2074
Construction of pYPQ141A, pYPQ141B, pYPQ141C and pYPQ141D:
2075
2076
2077
2078
2079
2080
2081
The AtU6 gRNA expression cassette was PCR amplified from AtU6-gBlock with primers gRNA-F and
gRNA-R. The PCR product was cloned into pYPQ104 (containing the attL5-attL2 cassette) at BamHI and
SpeI sites. The AtU3-gBlock was digested with BamH1 and SpeI and cloned to replace AtU6 gRNA
expression cassette in pYPQ141B, which lead to pYPQ141B. The OsU6-gBlock and OsU3-gBlock were
digested with BamH1 and EcoRI. The digested DNA fragments were cloned into pYPQ141A by replacing
the AtU6 gRNA expression cassette at BamHI and EcoRI sites, resulting in pYPQ141C and pYPQ141D
respectively.
2082
2083
2084
2085
2086
Construction of pYPQ142A, pYPQ143A, pYPQ144A, pYPQ145A, pYPQ146A, pYPQ147A and pYPQ148A:

The LacZ gene was PCR amplified from pFusA (Cermak et al. 2011) with primers pYPQ142-F and
pYPQ142-R and cloned into BamH1 and SpeI sites of pYPQ104 to make pYPQ142A. The other plasmids
were made similarly except these primers were used to pair with pYPQ142-F for PCR: pYPQ142-R,
pYPQ142-R, pYPQ142-R, pYPQ142-R, pYPQ142-R, pYPQ142-R and pYPQ142-R.
2087
2088
Construction of pYPQ131A, pYPQ132A, pYPQ133A, pYPQ134A, pYPQ135A, pYPQ136A, pYPQ137A and

pYPQ138A:
2089
2090
2091
2092
2093
2094
2095
The AtU6 gRNA expression cassette was PCR amplified from AtU6-gBlock with primers pYPQ131-F and
pYPQ131R which added BsaI sites to both ends of the PCR product. The PCR product was then cloned
into vector pHD1 (Cermak et al. 2011) at the two BsaI sites, resulting in pYPQ131A. Similarly, pYPQ132A,
pYPQ133A, pYPQ134A, pYPQ135A, pYPQ136A, pYPQ137A and pYPQ138A were made with
corresponding primers (pYPQ132-F, pYPQ132-R, pYPQ133-F, pYPQ133-R, pYPQ134-F, pYPQ134-R,
pYPQ135-F, pYPQ135-R, pYPQ136-F, pYPQ136-R, pYPQ137-F, pYPQ137-R, pYPQ138-F and pYPQ138-R)
and cloned to the BsaI sites of pHD2, pHD3, pHD4, pHD5, pHD6, pHD7 and pHD8 respectively.
2096
Construction of pYPQ131B, pYPQ132B and pYPQ133B:
2097
2098
2099
2100
The AtU3 gRNA expression cassette was PCR amplified from AtU3-gBlock with these three primer sets:
pYPQ131B-F and pYPQ131B-R, pYPQ132B-F and pYPQ132B-R, pYPQ133B-F and pYPQ133B-R. The
corresponding PCR fragments were cloned into pHD1, pHD2 and pHD3 at the two BsaI sites, resulting in
pYPQ131B, pYPQ132B andpYPQ133B.
2101
Construction of pYPQ131C, pYPQ132C and pYPQ133C:
2102
2103
2104
2105
The OsU6 gRNA expression cassette was PCR amplified from OsU6-gBlock with these three primer sets:
pYPQ131C-F and pYPQ131C/D-R, pYPQ132C-F and pYPQ132C/D-R, pYPQ133C-F and pYPQ133C/D-R. The
pYPQ131C, pYPQ132C andpYPQ133C.
2106
Construction of pYPQ131D, pYPQ132D and pYPQ133D:
Construction of vectors in the CRISPR/Cas9 toolkit:
47
2107
2108
2109
2110
The OsU6 gRNA expression cassette was PCR amplified from OsU6-gBlock with these three primer sets:
pYPQ131D-F and pYPQ131C/D-R, pYPQ132D-F and pYPQ132C/D-R, pYPQ133D-F and pYPQ133C/D-R. The
pYPQ131D, pYPQ132D andpYPQ133D.
2111
Construction of pYPQ150 (pcoCas9):
2112
2113
2114
2115
To construct pYPQ150, a previously-published plant-codon optimized version of Cas9 (pcoCas9) (Li et al.
2013) was amplified using primers pcoCas9-F1 and pcoCas9-R2. The PCR amplicons were digested with
NcoI and AatII and cloned into pNJB91, which contains attL1 and attR5 Gateway recombination sites
(Baltes et al. 2014).
2116
Construction of pYPQ151 (pcoCas9D10A):
2117
2118
2119
To construct pYPQ151, sequence from pcoCas9 was amplified in two separate PCR reactions using
primer pcoCas9-F1 and pcoCas9-R2, and pcoCas9-Gibson-F2 and pcoCas9-R1. The two amplicons were
then cloned into pNJB91 using Gibson assembly (Gibson et al. 2009).
2120
Construction of pNJB185 (pco-dCas9):
2121
2122
2123
2124
To construct pNJB185 (pco-dCas9; Cas9D10A H840A), sequence from pcoCas9 was amplified in three
separate PCR reactions using primer pair pcoCas9-F1 and pcoCas9- R2, and primer pair pcoCas9-F2 and
pcoCas9- R3, and primer pair pcoCas9-F3 and pcoCas9- R1. The three amplicons were then cloned into
pNJB91 using Gibson assembly, resulting in pNJB185.
2125
Construction of pYPQ152 (dCas9-VP63) and pYPQ-153 (dCas9-3X(SRDX)):
2126
2127
2128
2129
2130
2131
2132
The vector pNJB185 which contains pco-dCas9 was sequentially digested by Esp3I and AatII. At the same
time, a short DNA linker was made by annealing two oligos: Linker1-F and Linker1-R. The linearized
vector was gel-purified and ligated with annealed linker1. The resulting vector was named pYPQ185linker1. The VP64 DNA sequence was PCR-amplified with Pfu polymerase from gBlock_YPQ1 as
template with primers VP64-F (SalI) and VP64-R (AatII). The 3X(SRDX) sequence was PCR amplified from
gBlock_YPQ2 with primers SRDX3-F (SalI) and SRDX3-R (AatII). These two PCR products were cloned into
pYPQ185-linker1 vector at SalI and AatII sites, resulting in pYPQ152 and pYPQ153.
2133
Construction of pYPQ154 (AteCas9):
2134
2135
2136
2137
2138
The vector pNJB185 was digested by NcoI and AatII, and the 3136 bp vector backbone was gel-purified.
At the same time, a short DNA linker was made by annealing two oligos: Linker2-F and Linker2-R. The
linearized vector backbone was ligated with annealed linker2, resulting in pNJB185-linker2. The 4154 bp
sequence containing AteCas9 was cut from pDe-Cas9 vector (Fauser et al. 2014) by NcoI and SacI, and
ligated into pNJB185-linker2 at the same sites. The resulting vector was named pYPQ154.
2139
Construction of pYPQ155 (AteCas9D10A):
2140
2141
2142
2143
The D10A mutation was introduced by PCR amplification of the pYPQ154 vector with primers pYPQ155-F
(D10A) and pYPQ155-R (D10A) with Pfu polymerase. The amplified PCR product was cleaned up and
transformed to E. coli DH5. Successful recombination through overhangs of the PCR product resulted in
the new vector pYPQ155.
48
2144
Construction of pYPQ158 and pYPQ159:
2145
2146
2147
2148
2149
2150
The vector pNJB185-linker2 was digested with NcoI and AatII, and the linearized vector was gel-purified.
At the same time, a short DNA linker was made by annealing two oligos: Linker6-F and linker6-R.
Ligation of this linker to linearized pNJB185-linker2 vector resulted in pNJB-linker6. Vectors PX459
(hSpCas9) and PX460 (hSpCas9D10A) (Cong et al. 2013) were digested with AgeI, EcoRI and XbaI (triple
digestion). The corresponding Cas9 bands (4281 bp and 4284 bp) were cloned into pNJB185-linker6 at
MfeI and EcoRV sites, resulting in pYPQ158 and pYPQ159 respectively.
2151
Construction of pYPQ167:
2152
2153
2154
2155
Cas9p was PCR amplified from pYLCRISPR-Cas9-P35S-H vector (Ma et al. 2015) with NEB Q5 polymerase
and primers mCas9-F and mCas9-R. The amplified PCR product was cleaned up and digested with NcoI
and XbaI. The digested PCR product was cloned into pYPQ154 at NcoI and XbaI sites to replace the
AteCas9 sequence, which resulted in the new vector pYPQ167.
2156
B.
2157
Construction of pMDC99-SynPro-GUS-intron:
2158
2159
2160
2161
2162
2163
2164
2165
First, a GUS coding sequence was PCR amplified and cloned into pcr8 vector by TA cloning. One pcr8GUS clone with the correction orientation and sequence was confirmed by sequencing. This vector was
then digested with MfeI and the linearized vector was gel-purified. At the same time, an 85 bp intron
was amplified from a template plasmid (Hou et al. 2010) by PCR with primers GUS-intron-5 and GUSintron-3. This intron was cloned into pcr8-GUS through homologous recombination, resulting in pcr8GUS-intron. Then, the gBlock DNA (pGExpress1) was cloned into pcr8-GUS-Intron vector at AflII and MfeI
sites, resulting in pcr8-SynPro-GUS-intron. Finally, pMDC99-SynPro-GUS-intron was made by a Gateway
recombination between pMDC99 (Curtis and Grossniklaus 2003) and pcr8-SynPro-GUS-intron.
2166
Construction of pMDC32-pcoCas9-NbFLS2-gR1-gR2:
2167
2168
2169
2170
2171
2172
2173
First, oligos corresponding to NbFLS2-gR1 (NiFls2-gR1-top and NiFls2-gR1-bottom) NbFLS2-gR2 (NiFls2gR2-top and NiFls2-gR2-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearilzed
pYPQ131A and pYPQ132A through ligation which resulted in pYPQ131A-NbFLS2-gR1 and pYPQ132ANbFLS2 (step 1). Then, pYPQ131A-NbFLS2-gR1, pYPQ132A-NbFLS2-gR2, and pYPQ142 were put together
in a single Golden Gate reaction (step 2). The resulting construct, pYPQ142-NbFLS2-gR1-gR2, was mixed
with pYPQ150 and pMDC32 (Curtis and Grossniklaus 2003) a MultiSite Gateway reaction to make
pMDC32-pcoCas9-NbFLS2-gR1-gR2 (step 3).
2174
Construction of pMDC32-pcoCas9-NbFLS2-gR1-gR2-NbBAK1-gR3:
2175
2176
2177
2178
2179
2180
First, oligos corresponding to NbBAK1-gR3 (NiBAK1-gR3-top and NiBAK1-gR3-bottom) were

phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ133A through ligation, resulting in
pYPQ133A-NbBAK1-gR3 (step 1). Then, pYPQ131A-NbFLS2-gR1, pYPQ132A-NbFLS2, pYPQ133A-NbBAK1gR3 and pYPQ143 were put together in a single Golden Gate reaction, which resulted in pYPQ143NbFLS2-gR1-gR2-NbBAK-gR3 (step 2). This gRNA entry clone was then mixed with pYPQ150 and pMDC32
in a MultiSite Gateway reaction to make pMDC32-pcoCas9-NbFLS2-gR1-gR2-NbBAK1-gR3 (step 3).
2181
Construction of pMDC32-pcoCas9-NbFLS2-gR1-gR2-gR3:
Construction of 15 T-DNA vectors:
49
2182
2183
2184
2185
2186
2187
First, oligos corresponding to NbFLS2-gR3 (NiFLS2-gR3-top and NiFLS2-gR3-bottom) were

phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ133A through ligation, resulting in
pYPQ133A-NbFLS2-gR3 (step 1). Then, pYPQ131A-NbFLS2-gR1, pYPQ132A-NbFLS2, pYPQ133A-NbFLS2gR3 and pYPQ143 were put together in a single Golden Gate reaction, which resulted in pYPQ143NbFLS2-gR1-gR2- gR3 (step 2). This gRNA entry clone was then mixed with pYPQ150 and pMDC32 in a
MultiSite Gateway reaction to make pMDC32-pcoCas9-NbFLS2-gR1-gR2--gR3 (step 3).
2188
Construction of pMDC32-pcoCas9-NbFLS2-gR4-gR5-gR6:
2189
2190
2191
2192
2193
2194
First, oligos corresponding to NbFLS2-gR4 (NiFLS2-gR4-top and NiFLS2-gR4-bottom), NbFLS2-gR5(NiFLS2gR3-top and NiFLS2-gR5-bottom) and NbFLS2-gR6 (NiFLS2-gR6-top and NiFLS2-gR6-bottom) were
phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A
respectively (step 1). These three vectors were then mixed with pYPQ143 for Golden Gate reaction to
make pYPQ143-NiFLS2-gR4-gR5-gR6 (step 2). Finally, this gRNA entry clone was mixed with pYPQ150
and pMDC32 in a MultiSite Gateway reaction to make pMDC32-pcoCas9-Nb-FLS2-gR4-gR5-gR6 (step 3).
2195
Construction of pMDC32-pcoCas9-NbFLS2-gR7 and gR9:
2196
2197
2198
2199
2200
2201
First, oligos corresponding to NbFLS2-gR7 (NiFLS2-gR7 (U3)-top and NiFLS2-gR7 (U3)-bottom) and
NbFLS2-gR9 (NiFLS2-gR9 (U3)-top and NiFLS2-gR9 (U3)-bottom) were phosphorylated and annealed, and
cloned into Esp3I-linearized pYPQ131B and pYPQ132B respectively (step 1). These two vectors were
then mixed with pYPQ142 for Golden Gate reaction to make pYPQ142-NiFLS2-gR7-gR9 (step 2). Finally,
this gRNA entry clone was mixed with pYPQ150 and pMDC32 in a MultiSite Gateway reaction to make
pMDC32-pcoCas9-Nb-FLS2-gR7-gR9 9 (step 3).
2202
Construction of pMDC32-pcoCas9-NbBAK1-gR1-gR2-gR3:
2203
2204
2205
2206
2207
2208
2209
First, oligos corresponding to NbBAK1-gR1 (NiBAK1-gR1-top and NiBAK1-gR1-bottom), NbBAK1-gR2

(NiBAK1-gR2-top and NiBAK1-gR2-bottom), and NbBAK1-gR3 (NiBAK1-gR3-top and NiBAK1-gR3-bottom)
were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and
pYPQ133A respectively (step 1). These three vectors were then mixed with pYPQ143 for Golden Gate
reaction to make pYPQ143-NiBAK1-gR1-gR2-gR3 (step 2). Finally, this gRNA entry clone was mixed with
pYPQ150 and pMDC32 in a MultiSite Gateway reaction to make pMDC32-pcoCas9-NbBAK1-gR1-gR2-gR3
(step 3).
2210
Construction of pMDC32-pcoCas9-NbBAK1-gR4-gR5-gR6:
2211
2212
2213
2214
2215
2216
2217
First, oligos corresponding to NbBAK1-gR4 (NiBAK1-gR4-top and NiBAK4-gR1-bottom), NbBAK1-gR5

(NiBAK1-gR5-top and NiBAK1-gR5-bottom), and NbBAK1-gR6 (NiBAK1-gR6-top and NiBAK1-gR6-bottom)
reaction to make pYPQ143-NiBAK1-gR4-gR5-gR6 (step 2). Finally, this gRNA entry clone was mixed with
pYPQ150 and pMDC32 in a MultiSite Gateway reaction to make pMDC32-pcoCas9-NbBAK1-gR4-gR5-gR6
(step 3).
2218
Construction of pMDC32-pcoCas9-OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U6U3U6):
2219
2220
2221
First, oligos corresponding to OsYSA-gR1 (YSA-gR1-OsU6-F and YSA-gR1-OsU6-R), ROC5-gR1 (ROC5-gR1OsU3-F and ROC5-gR1-OsU3-R) and OsYSA-gR2 (YSA-gR2-OsU6-F and YSA-gR2-OsU6-R) were
phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131C, pYPQ132D and pYPQ133C
50
2222
2223
2224
2225
respectively (step1). These three vectors were then mixed with pYPQ143 for Golden Gate reaction to
make pYPQ143-OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U6U3U6) (step 2). Finally, this gRNA entry clone was
mixed with pYPQ150 and pMDC32 in a MultiSite Gateway reaction to make pMDC32-pcoCas9-OsYSAgR1-ROC5-gR1-OsYSA-gR2 (U6U3U6).
2226
Construction of pMDC32-pcoCas9-OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U3U6U3):
2227
2228
2229
2230
2231
2232
2233
First, oligos corresponding to OsYSA-gR1 (YSA-gR1-OsU3-F and YSA-gR1-OsU3-R), ROC5-gR1 (ROC5-gR1OsU6-F and ROC5-gR1-OsU6-R) and OsYSA-gR2 (YSA-gR2-OsU3-F and YSA-gR2-OsU3-R) were
phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131C, pYPQ132D and pYPQ133C
respectively (step1). These three vectors were then mixed with pYPQ143 for Golden Gate reaction to
make pYPQ143- OsYSA-gR1-ROC5-gR1-OsYSA-gR2 (U3U6U3) (step 2). Finally, this gRNA entry clone was
mixed with pYPQ150 and pMDC32 in a MultiSite Gateway reaction to make pMDC32-pcoCas9-OsYSAgR1-ROC5-gR1-OsYSA-gR2 (U3U6U3).
2234
Construction of pYPQ202-pco-dCas9-VP64-AtPAP1-gR1-gR2-gR3:
2235
2236
2237
2238
2239
2240
2241
First, oligos corresponding to AtPAP1-gR1 (PAP1-gR1-top and PAP1-gR1-bottom), AtPAP1-gR2 (PAP1gR2-top and PAP1-gR2-bottom), and AtPAP1-gR3 (PAP1-gR3-top and PAP1-gR3-bottom) were
phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A
respectively (step 1). These three vectors were then mixed with pYPQ143 for Golden Gate reaction to
make pYPQ143- AtPAP1-gR1-gR2-gR3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ152
and pYPQ202 (containing an Arabidopsis UBQ10 promoter) in a MultiSite Gateway reaction to make
pYPQ202-pco-dCas9-VP64-AtPAP1-gR1-gR2-gR3 (step 3).
2242
Construction of pYPQ202-pco-dCas9-VP64-miR319-gR1-gR2-gR3:
2243
2244
2245
2246
2247
2248
2249
First, oligos corresponding to miR319-gR1 (miR319-gR1-top and miR319-gR1-bottom), miR319-gR2

(miR319-gR2-topand miR319-gR2-bottom), miR319-gR3 (miR319-gR3-top and miR319-gR3-bottom)
reaction to make pYPQ143- miR319-gR1-gR2-gR3 (step 2). Finally, this gRNA entry clone was mixed with
pYPQ152 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-pco-dCas9-VP64- miR319gR1-gR2-gR3 (step 3).
2250
Construction of pYPQ202-pco-dCas9-VP64-AtFIS2-gR1-gR2-gR3:
2251
2252
2253
2254
2255
2256
First, oligos corresponding to AtFIS2-gR1 (FIS2-gR1-top and FIS2-gR1-bottom), AtFIS2-gR2 (FIS2-gR2-top

and FIS2-gR2-bottom), AtFIS2-gR3 (FIS2-gR3-top and FIS2-gR3-bottom) were phosphorylated and
annealed, and cloned into Esp3I-linearized pYPQ131A, pYPQ132A and pYPQ133A respectively (step 1).
These three vectors were then mixed with pYPQ143 for Golden Gate reaction to make pYPQ143- AtFIS2gR1-gR2-gR3 (step 2). Finally, this gRNA entry clone was mixed with pYPQ152 and pYPQ202 in a
MultiSite Gateway reaction to make pYPQ202-pco-dCas9-VP64- AtFIS2-gR1-gR2-gR3 (step 3).
2257
Construction of pYPQ202-pco-dCas9- 3X(SRDX)-AtCSTF64-gR1-gR2-gR3:
2258
2259
2260
2261
First, oligos corresponding to AtCSTF64-gR1 (CSTF64-gR1-top and CSTF64-gR1-bottom), AtCSTF64-gR2

(CSTF64-gR2-top and CSTF64-gR2-bottom), and AtCSTF64-gR3 (CSTF64-gR3-top and CSTF64-gR3-bottom)
51
2262
2263
2264
reaction to make pYPQ143- AtCSTF64-gR1-gR2-gR3 (step 2). Finally, this gRNA entry clone was mixed
with pYPQ153 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202-pco-dCas9- 3X(SRDX)AtCSTF64-gR1-gR2-gR3 (step 3).
2265
Construction of pYPQ202-pco-dCas9- 3X(SRDX)- miR159A-gR2-miR159B-gR1-gR2:
2266
2267
2268
2269
2270
2271
2272
First, oligos corresponding to miR159B-gR2 (miR159B-gR2-top and miR159B gR2-bottom), miR159AgR2 (miR159A-gR2-top and miR159A-gR2-bottom), and miR159B-gR1 (miR159B-gR1-top and miR159B gR1-bottom) were phosphorylated and annealed, and cloned into Esp3I-linearized pYPQ131A,
pYPQ132A and pYPQ133A respectively (step 1). These three vectors were then mixed with pYPQ143 for
Golden Gate reaction to make pYPQ143- miR159A-gR2-miR159B-gR1-gR2 (step 2). Finally, this gRNA
entry clone was mixed with pYPQ153 and pYPQ202 in a MultiSite Gateway reaction to make pYPQ202pco-dCas9- 3X(SRDX)-miR159A-gR2-miR159B-gR1-gR2 (step 3).
2273
Sequence and annotation of target genes and gRNA sites:
2274
2275
2276
2277
Note: 3 PAM sequences are underlined. All gRNAs were either designed with the CRISPR-P program (Lei
et al. 2014) or selected manually. In some occasions, an extra G or A nucleotide was added to gRNA 5
ends when the first nucleotide was not G or A in order to accommodate U6 or U3 promoter
requirements. The gRNAs are color-coded for indication of different orientations.
2278
>NbFLS2-partial genomic
2279
2280
2281
2282
2283
2284
2285
2286
2287
2288
2289
2290
2291
2292
2293
2294
2295
2296
2297
2298
2299
2300
ATGTCACAGACAGTTTTATATGCATTAGCCATATTTTCCTTTACCTTTTTCATACCTTTGTCATATGGCCAAACTCCAA
GCTTGGAAGTTGAAGTTGCTGCTTTAAAAGCTTTCAAGAACTCAATCTCTGATGACCCCTATGGTGCACTTGTGGAT
TGGACTGATGCAAATCACCATTGCAACTGGTCTGGAATTACATGTGATCCTTCTTCAAACCATGTCATCAACATTAC
ACTGTTTGAGACGCAGCTCAAAGGGGAAATCTCTCCGTTTCTGGGAAACCTCTCCAAACTCCAGGTTCTTGATCTA
ACTTTGAATTCATTTACTGGAAATATTCCACCTCAGCTGGTGCATTGCACAGAGCTGGTTGAGCTCATTCTTTATGA
AAATTCTCTTTTTGGTAAAATTCCTGCTGAACTCGGAAACCTCAAAAACCTGCAGTTAATAGATTTTGGAAATAACT
TTCTGAATGGGAACATACCTGAGAGTATATGCAAGTGCACTGAATTGTTGTTAGTAGGCCTCATCAACAACAGTTT
CACAGGCAAATTACCGTCTGATATTGGTAATTTGGCTAATCTTCAGTTGTTTGTAGCTTATGAAAACAATCTCATTG
GTTCTATTCCTATCTCCATTGGAAAGTTAACAGCTTTGCAAGCCCTTGATCTGAGCGAAAACCAATTGTCTGGACCT
ATACCACCAGAAATTGGCAACTTGTCAAGTTTAGAAACTCTTCACTTGCATCTCAACTTTCTTTCTGGTAAAATTCCT
TCTGAACTTGGCCTCTGTACCAATCTTGTTACCTTGAACATGTATACTAATCACTTCACTGGAAGCATACCTACTGA
GCTTGGAAATTTAGAAAACTTGCAAACGCTCAGATTGTATGACAATCAGTTGAACTCCAGTGTACCACCCTCATTGT
TCCACCTGAAGTCATTAGCTCATTTAGGACTCTCACAGAATGAGCTAACTGGCCAAATTCCTCCCGAGTTGGGATCT
TCAATGTCACTACAAGTGCTTACCCTCCACTCAAATAGGTTGTCCGGGGAAATTCCATCAACTATAACAAAACTGAC
AAACTTGACATATTTGTCTTTGAGCTTCAATTTATTGACCGGATCACTTCCATTAGAATTTGGGTTGCTCTATAACCT
GAAGAATCTAACTGCAAGTAACAACCTCCTAGAGGGATCTATTCCATCTAGCATAATAAATTGTTCACATCTTCTAG
TTTTGACCCTCTCTTATAATAAAATAACAGGGGAAATACCCATTGGATTGGGGCAGTTGTCCAATCTTACATTTTTG
TCTTTGGGATCAAACAAAATGGCGGGGGAGATTTCTGACGATTTCTTCAACTGCTCAATGCTTGAAGTCCTAGATC
TGAGTGATAACAATTTCAGTGGGAAACTCAAACCGATGATTGGCAGACTTTCTAAACTTCGAGTTCTAAGAGCCCG
TTCTAATTCCTTTCTTGGGCCAATCCCACCAGAGATTGGTAAACTGAGTCAACTGATGGATTTAGTGCTTGACGAAA
ACAGTTTCTCAGGTGTGATACCACCAGAGATTTCAATGCTTTCAGGCCTCCAGGGCCTTTGGCTGTCTGACAACAA
GCTGGAAGGTAAACTTCCGGTGCAACTTTTTGAGCTTAAAAAACTCAATGAACTCCGGCTGCAGAACAATAATTTC
52
2301
2302
2303
2304
TTCGGTCCAATACCTCACCAGATATCCAAACTGGAGTTACTCTCGTTGCTGGATCTGAGCAGAAACAAGCTTAATG
GTACAATCCCAGAGAGCATGGCGAGCCTCCGCAGACTGATGACCTTAGATCTTTCACACAACCTTCTTACTGGATC
CGTTCCTCGAGCAGTACTCGCCAGCATGAGAAGTATGCAATTTTACTTGAAGGTTTCCAGCAACTTTTTGGATGGA
ACGATCCCAGATGAGATTGGCGTGTTAGAAATGGTTCAAGAGATTGATATGTCAAACAACAATCTATCAGGCAGC
2305
>NbBAK1-partical genomic
2306
2307
2308
2309
2310
2311
2312
2313
2314
2315
2316
2317
2318
2319
2320
2321
2322
2323
2324
2325
2326
2327
2328
2329
2330
2331
2332
2333
2334
2335
2336
2337
2338
2339
2340
GTTCTTTTCTGAATTGAAGGAGGCTGAATAATAACAGTTTGAGTGGACGTATTCCCATGTCTCTAACCACCATTCTT
GTACTTCAAGTACTGTAAGTGTAAAGTTGTTCCCTTATTTATCAAATGCTGTCTGTTGTCCGTGTTGGTCTACTCTTT
TGGGTAACTAATTTTCATTTCCTTGCAATGCAGTGATCTCTCAAGCAATCATTTGACAGGACCAGTTCCAGTCAATG
GTTCCTTTTCGCTTTTTACTCCTATAAGGTGTGTCAGAAGCACAAGTTTGTTGTTACTTTCTGTTCCTGAAGTAACAA
AAGTGATTATCTAACTATAGAGTACCTATATTTCAGTTTTGCTAATAACCAATTGGAAGTTCCTCCAGCTTCTCCGCC
TCCGCCTCTTCCTCCTACACCCTCATCTTCTTCTTCAGGTTTGTTGAGATTATTTGTGTTGATACTGTTATGAAACTGT
GCTACCTCTCCTTTTTCCCTTTTCCAATTTAGTTTTTGTGAAAAGAACCCTTTAGATTATTCACGAGAAGGGGAGTAG
GCTATGGGGAAAAAGGAAAGGCCTGGTTGCCTGGGGAAGAGAACAAACAAGATTCCCTTGTGAAAGCAGTTACA
AATAAAAGCTATTATTACTTTAGGCACTAATAAATATTAGCTTTTTTAGATCAGTTGTACAGGTGTTGGAGTCTTGT
TGTAATCTAGCACGCATGACTTTTAAACTAAACCTTTCAACTCTGCATCTTTTTTTCTTTTTTCGGAAATTAGATTTAC
TGTAAACTATGAAATGTGAGTGGTGGTGTGTCAAAAGTACCTTATATTATTTAGAGTTGCTTGTATATTTAAATTCA
GGTCATACAAATAAAAAGTTAAATCAGCTTATGTATTGGAAACTCGATCATCTTTTCAGTGGGCAACAGTGCAACT
GGAGCCATCGCTGGAGGAGTTGCTGCAGGCGCTGCTCTTCTATTTGCAGCTCCTGCAATTTTTCTTGCTTGGTGGC
GTCGGAGGAAACCACAAGATCACTTCTTTGATGTTCCTGGTTCGGACACTCAAACTGAAATTTGAGTTAATTGTACT
TTCCACTTGATGTGTTCTTACTCAGTTATTATAACCTCTTTTTGCAGCTGAGGAGGATCCAGAAGTTCATCTAGGAC
AACTCAAGAGGTTTTCCTTGCGTGAACTACAAGTTGCATCAGATAATTTTAGCAACAAAAATATACTTGGTAGAGG
TGGATTTGGTAAGGTTTATAAGGGCCGGTTAGCTGATGGCTCTTTAGTTGCAGTGAAAAGACTAAAAGAGGAACG
TACTCAAGGTGGGGAGTTACAGTTTCAGACAGAAGTAGAAATGATCAGCATGGCTGTACACCGAAATCTACTTCG
TTTAAGGGGCTTTTGCATGACTCCCACTGAGCGCGTGCTTGTTTATCCATACATGGAGAACGGAAGTGTGGCATCA
CGTTTAAGAGGTATCTCTTGTTTTTTTTTAATTTTCCTGGACTTGCCTCTAGCTTTTGCTCACTTTTAGTCAGGCGAAA
TGGATCAAACAAAGAAAAAGCCAATAGCTACTCAAAATGATAAGCCTGTGATCTTTAATTTCTCTTTCATAAATTTG
TAACAGGTTTGAAGAAGTTACTGCAGAAGTGGAAGCCAATAGCTATTTGATTTGTTAGTGGTTGAGTCTTTTATGA
CTTCTCATGATGGACATGTATATTTAACAAGTCGATGTCGTCTTATGATTTTGCTAGTTTCTATGATTCTATCAACCA
ATTTCACAAGTGGTGCTATCCTTGATATCTTACTGAATATTAAAGATTTGAGTTGCTGATTAAGATGTATTTTATGAT
CACATTTGTAAGTCAACCAAACAATTAGATATCTTAAAAAAGAGAATAAATTTTATTATTGTATGAAGCTGGAGAT
AATGATGCCTTATAACTTGGAATGGGAGCCTTGGAGCAACGATAAAGTTGTCTCTGTGTGACCTATAGGTCACGG
GTTCGAGCGGTGGAAGAAACCCACTAATTCTCGCAATAGGGTAGGTTGTCTACATCGCATTCCTTGGGGTATGGCC
CTTCCTCCGACCCTGCATGAACGCGAGATGCCTTGTGCATTGGCCTGCCCTTTTTAAAGTTATATTGACCTGAAAAG
ATCAGTTTTTCTTCTAAAAACTTGTTCAAGCTTTTCCTCGGAACCTGTTTCCAGTTCACGTAATAGCTTGAATTTATTC
TCATGGATGATTTACATTCTTGTAGAGCGGCCTGAATCGGAGCCCCCACTTGACTGGCCAAAAAGGAAGCGTATTG
CACTTGGGTCGGCAAGAGGCCTTGCTTACTTGCATGATCATTGTGATCCTAAGATTATTCATCGTGATGTCAAAGCC
GCAAATATCTTATTGGATGAGGATTTTGAAGCAGTTGTTGGGGATTTTGGGTTAGCTAAACTCATGGACTACAAGG
ATACTCACGTTACCACTGCTGTACGTGGTACAATTGGACATATTGCCCCTGAATATTTATCCACTGGTAAATCTTCT
GAGAAGACGGATGTGTTTGGCTATGGGGTTATGCTTCTAGAGCTCATAACTGGCCAAAGGGCTTTTGATCTTGCTC
GACTTGCGAATGATGATGATGTCATGTTGCTTGATTGGGTAAGCAT
53
2341
2342
>OsYSA
2343
2344
2345
2346
2347
2348
2349
2350
2351
2352
2353
2354
2355
2356
2357
2358
2359
2360
2361
2362
2363
2364
2365
2366
2367
2368
2369
2370
2371
2372
ATGCCCCGCGTTTGCGCCGCCCCTCGGGCGCCGCCGCCGCCGTGCCCGTGCCATGTCGGAGTAGGGCCGCTTCGG
CCGAGGTGGCGCGCCTCCCGGCACGGCCCTCTCCGGGCGGCTGGCCAGGAGCAGCTCCTCACCGCCCTGCGCGA
GCAGCCGGACCCCGACGCGGCGCTCCGGATGCTCAACGCGGCGCTGGCGCGGGACGACTTCGCGCCCGGCCCCG
AGGTCTACGAGGAGATCATCCGCAAGCTCGGCGCGGTCGGGGCCCTCGACCTCATGAAGGTGCTCGTCGCGGAG
ATGCGGCGGGAGGGGCACCAGGTGAAATTGGGCGTAGTCCACTCCTTCTTGGACAGCTACGAGGGGCAGCAGCT
GTTCGACGATGCCGTCGACCTGATTCTGAATCAACTCCAACCATTGTTTGGCATTCAGGCAGACACCGTGGTGTAC
AATCACCTTCTCAATGTTCTTGTGGAGGGGAGCAAAATGAAACTCCTTGAATCAGTGTACTCGGAGATGGGTGCTA
GGGGAATCAAGCCTGATGTTGTCACATTCAACACACTGATGAAGGCGTTGTGCCGAGCACATCAGGTCAGGACTG
CAGTTCTCATGCTCGAGGAAATGTCTAGCAGAGGCGTGGCGCCTGACGAGACGACGTTTACCACCCTGATGCAAG
GATTTGTCGAGGAGGGGAGCATCGAGGCTGCACTGAGGGTCAAAGCCAGGATGTTGGAAATGGGGTGCTCGGC
GACGAAGGTGACGGTTAATGTTTTGATTAATGGGTACTGCAAGCTAGGGAGGGTGGAGGATGCTCTTGGGTATAT
ACAGCAGGAGATTGCCGATGGGTTTGAGCCTGACCAGATCACATATAACACTTTTGTTAATGGTCTCTGCCAAAAT
GATCATGTCGGCCATGCCCTCAAAGTCATGGATGTGATGGTTCAGGAGGGCCATGATCCTGATGTTTTCACCTACA
ATATCGTTGTGAATTGCCTTTGTAAAAATGGACAGCTTGAAGAGGCAAAAGGAATTCTGAATCAGATGGTGGATC
GGGGTTGTTTGCCTGACATTACCACATTCAACACTCTCATTGCTGCCTTATGCACGGGGAATCGACTTGAGGAAGC
CTTGGACCTTGCACGTCAGGTTACAGTGAAGGGAGTCTCTCCAGATGTTTATACTTTCAATATTCTGATTAACGCGC
TCTGCAAAGTAGGCGATCCTCATCTTGCACTTCGATTGTTTGAAGAGATGAAGAACAGTGGATGCACCCCGGATG
AAGTAACATACAATACTTTGATTGACAATCTTTGCTCACTTGGGAAGCTTGGTAAAGCATTGGATTTGTTAAAAGAT
ATGGAGTCCACTGGTTGTCCTCGAAGTACAATTACATATAACACTATAATTGACGGGTTATGCAAGAAAATGAGAA
TAGAAGAAGCAGAAGAAGTTTTTGATCAAATGGATCTGCAAGGCATTTCGAGGAATGCAATCACATTCAATACTCT
CATCGATGGTTTGTGCAAGGACAAAAAGATTGATGATGCTTTTGGGCTTATTAATCAAATGATAAGTGAAGGGTT
GCAACCTAACAATATCACTTACAATTCTATTCTAACTCATTATTGCAAGCAAGGTGACATAAAAAAGGCTGCGGAT
ATTTTAGAAACTATGACTGCAAATGGATTTGAAGTGGATGTTGTTACGTACGGTACTCTGATTAACGGTCTATGCA
AGGCTGGTAGGACACAGGTTGCTTTGAAGGTACTCAGAGGTATGCGGATAAAAGGGATGAGGCCTACTCCAAAA
GCTTACAATCCTGTGCTCCAGTCTCTCTTCAGACGGAATAATATCAGAGATGCCCTGAGTCTTTTCAGGGAGATGG
CAGAGGTTGGTGAGCCTCCTGATGCTTTGACATATAAGATTGTTTTTCGTGGGCTCTGTCGTGGTGGAGGGCCTAT
TAAAGAAGCTTTTGATTTCATGTTGGAGATGGTTGATAAGGGGTTCATACCAGAGTTCTCATCCTTCCGTATGCTAG
CTGAAGGTCTATTAAACCTGGGTATGGATGATTACTTCATTAGAGCCATTGAAATAATCATGGAAAAGGTCGACCT
CAGAGAGTCTGATGTTTCTGCAATAAGGGGATATCTCAAGATCCGCAAATTTTATGATGCATTAGCAACCTTTGGC
CGTTTCCTGGAGATCAACAACCCTCAATGGAGTTACCGATGA
2373
>OsROC5
2374
2375
2376
2377
2378
2379
2380
2381
2382
2383
2384
2385
ATGCAGTTCCCTTTCGCCAGTGGCTTCGCGTCCTCGCCGGCGCTCTCCCTCGCGCTGGACAACGCCGGCGGTGGCA
TCGGCGGGCGGATGCTCGGCGGCGGCGCTGGCGCTGGTTCTAGCGCGGGCGGCGCGATGACGAGGGACACTGA
GGCGGAGAACGACAGCCGGTCGGGAAGCGACCATCTCGACGCCATCTCGGCCGCCGGCGAGGACGACGTCGAG
GACGCCGAGCCCAGCAACTCCCGCAAGAGGAAGAAGCGATACCACCGCCACACGCCGCAGCAGATCCAAGAGCT
CGAAGCGCTGTTCAAGGAGTGCCCCCATCCGGACGAGAAGCAGCGCGCCGAGCTCAGCAGGAGGCTCTCCCTCG
ATGCGCGGCAGGTCAAGTTCTGGTTCCAGAATCGCCGCACGCAGATGAAGACGCAACTGGAGCGGCACGAGAAC
GCGCTTCTCAAGCAGGAGAACGACAAGCTGCGCGCCGAGAACATGACGATCCGGGAGGCGATGCGCAGCCCAAT
GTGCGGCAGCTGCGGGAGCCCCGCCATGCTCGGGGAGGTGTCCCTAGAGGAGCAGCACCTGCGCATCGAGAATG
CGCGCCTCAAGGACGAGCTCAACCGCGTGTGCGCCCTCGCCACCAAGTTCCTAGGCAAGCCCATCTCCCTCCTGTC
GCCGCCGCCCTTGCTGCAGCCGCACCTCTCGCTGCCTATGCCTAACTCGTCGCTGGAGCTAGCGATCGGAGGCATC
GGTGGCTTAGGGTCTCTCGGTACACTACCCGGCTGTATGAATGAGTTCGCCGGAGGCGTGTCGAGCCCGATGGGC
ACGGTGATCACGCCTGCGCGCGCAACCGGGGCTGCTATACCATCGCTGGTGGGTAACATCGACAGGTCCGTGTTC
54
2386
2387
2388
2389
2390
2391
2392
2393
2394
2395
2396
2397
2398
2399
2400
2401
2402
2403
2404
2405
TTAGAGCTTGCAATCAGCGCGATGGACGAACTTGTCAAGATGGCACAGATGGACGATCCATTGTGGGTTCCGGCA
CTGCCTGGTTCTCCCAGCAAGGAGGTGCTCAACTTTGAGGAGTATCTTCACTCCTTCCTACCGTGCATTGGCATGAA
GCCTGCCGGCTACGTCTCTGAGGCCTCGAGGGAATCCGGTCTAGTCATCATCGACAACAGCCTTGCCCTTGTAGAG
ACCCTCATGGATGAGAGACGGTGGTCTGACATGTTCTCGTGCATGATTGCTAAGGCAACAGTGCTTGAGGAGGTG
TCTACCGGCATTGCAGGAAGCAGAAATGGCGCGTTGCTGCTGATGAAGGCTGAGCTACAGGTGCTCTCACCTCTA
GTACCAATTAGGGAGGTTACATTTCTCCGGTTTTGTAAGCAGCTGGCTGAGGGTGCATGGGCAGTAGTTGATGTG
TCCATTGATGGTTTAGTGAGAGATCATAATTCTGGAACGGCACCCACAGGTGGAAATGTGAAGTGTAGGAGGGTA
CCTTCCGGCTGTGTGATGCAAGACACTCCCAATGGGTATTGCAAGGTCACATGGGTTGAGCATACGGAATACGAT
GAGGCATCAGTGCACCAGCTCTACCGTCCACTCCTTCGGTCTGGTCTCGCCTTTGGCGCCAGGCGTTGGCTTGCGA
CGCTGCAGCGCCAATGCGAATGCCTTGCCATCCTCATGTCCTCTGCTACAGTGACAGCAAATGACTCGACTGCTAT
ATCGCAAGAGGGCAAGCGAAGCATGCTGAAGCTGGCACGCCGGATGACGGAGAACTTCTGTGCTGGGGTGAGC
GCATCGTCTGCACGTGAATGGAGCAAGCTGGATGGTGCGACCGGTAGCATCGGGGAGGACGTGCGCGTGATGGC
ACGGAAGAGCGTGAGCGAGCCTGGAGAGCCACCGGGCGTGGTGCTGAGCGCGGCCACCTCGGTGTGGGTGCCC
GTGGCTCCGGAGAAGCTCTTCAACTTCCTGCGCGATGAGCAGCTGCGTGCGGAGTGGGACATCCTCAGCAATGGA
GGCCCCATGCAGGAGATGACCCAGATCGCCAAGGGGCAGCGGGATGGGAACTCGGTTTCACTCCTCAGGGCCAG
TGCTGTGAGTGCCAACCAGAGCAGCATGCTGATACTTCAGGAGACGTGCACCGACGCTTCTGGCTCCATTGTTGTG
TACGCTCCGGTAGACATCCCGGCAATGCAGCTTGTCATGAATGGTGGCGACTCCACCTATGTTGCTCTGCTTCCATC
CGGATTCGCCATCCTGCCCGATGGGCCTCGCATCGGCGCCACCGGGTACGAGACGGGCGGCTCTCTGCTCACCGT
GGCGTTCCAGATTCTTGTCAACAACCAGCCCACCGCCAAGCTCACCGTGGAATCGGTCGAGACCGTGAACAATCTC
ATCTCCTGCACCATCAAGAAGATCAAGACGGCGCTGCAGTGCGACGCCTGA
2406
>AtPAP1 (At1g56650)
2407
2408
2409
2410
2411
2412
2413
2414
2415
2416
2417
2418
2419
2420
2421
2422
2423
2424
2425
2426
2427
2428
ctctatccaaactacacggatgaagcttattgttattcatccaccctttttctcaattctgtcctatttcttgtgcatgaaacttctccatcttgtaatcggat
aaatcatacccaaattttttctttctgaaaacatatatacccgaacattaattactatcgtcctttctcctaattttgttaagaaacatgtttgtttgttttta
gtactgaaaaaggatggagatacttgctagatcctatgaaccttttctctctaggacaaatcagtaaccaaacaataacttagcaaattaagcacgac
agctaatacataaaatgtggatatcaaacatgcacgtcacttccttttttccgtcacgtgtttttataaattttctcacatactcacactctctataagacc
tccaatcatttgtgaaaccatactatatataccctcttccttgaccaatttacttataccttttacaatttgtttatatattttacgtatctatctttgttccAT
GGAGGGTTCGTCCAAAGGGCTGCGAAAAGGTGCTTGGACTACTGAAGAAGATAGTCTCTTGAGACAGTGCATTA
ATAAGTATGGAGAAGGCAAATGGCACCAAGTTCCTGTAAGAGCTGgtatgttatttacgaacacacacacactaaccgacacac
acacacacaaatatgaatatctataatcactaccaatagtcttcgttctctctattttctattcagaaaattgattaatacccggtattaaaaaaaaaaa
aaaaaatttgtttaaatgagtacaaatcattgttacaacttctttatgctgtttttacatgctattaaaggttgtgcatgaaaatttcttttgctgttcgtatt
tgttttacacctaaacgaagatttttacttaaaattaaagaaaaaaaattatactaattttagttacgttgcgtattgctagcttctcctataaagtcgttc
aaatttttacacgcttgtcttcttgtaaatgaattcgtgggaaaattttgtatgaacacgtgtttctgtgttggaacagttctttatttttattggtgtgcata
gattcttcctgataaaatatatagaaggagacaaataaaaaacagtcttagtatgtaggtataatcaaagaatcaattattggttttgtagGGCTAA
ACCGGTGCAGGAAAAGTTGTAGATTAAGATGGTTGAACTATTTGAAGCCAAGTATCAAGAGAGGAAAACTTAGCT
CTGATGAAGTCGATCTTCTTCTTCGCCTTCATAGGCTTCTAGGGAATAGgtattaattgttacctcgatactacttaactcggaga
gtcgtcataagttaatactaataacatatgtatattttcttacaattgttagGTGGTCTTTAATTGCTGGAAGATTACCTGGTCGGACC
GCAAATGACGTCAAGAATTACTGGAACACTCATCTGAGTAAGAAACATGAACCGTGTTGTAAGATAAAGATGAAA
AAGAGAGACATTACGCCCATTCCTACAACACCGGCACTAAAAAACAATGTTTATAAGCCTCGACCTCGATCCTTCAC
AGTTAACAACGACTGCAACCATCTCAATGCCCCACCAAAAGTTGACGTTAATCCTCCATGCCTTGGACTTAACATCA
ATAATGTTTGTGACAATAGTATCATATACAACAAAGATAAGAAGAAAGACCAACTAGTGAATAATTTGATTGATGG
AGATAATATGTGGTTAGAGAAATTCCTAGAGGAAAGCCAAGAGGTAGATATTTTGGTTCCTGAAGCGACGACAAC
AGAAAAGGGGGACACCTTGGCTTTTGACGTTGATCAACTTTGGAGTCTTTTCGATGGAGAGACTGTGAAATTTGAT
TAG
55
2429
>miR319 (At4g23713)
2430
2431
2432
2433
2434
2435
2436
2437
2438
Agtgtatttcctcgcatctaccatcccttttctacgcctctctccctctctctctttctccatcaaatcttgttttgttcaaactctctctctctcatctattctct
ccatacaatacatgaatatacatacataccatcatcttcttttcccatctctagtttttcatcaatcttctgatgttccaaacgctctatctcttcatatacat
acatacgaatatattattgttgtcatagatccatttagaatcactttagcttttagatgagatctagggtttctttgttttctttcaaattttgttgcatattct
tctaaatcatggtttttcgcttgctaggttatagatccatgcaaatatggagtagatgtacaaacacacgctcggacgcatattacacatgttcatacac
ttaatactcgctgttttgaattgatgttttaggaatatatatgtAGAGAGAGCTTCCTTGAGTCCATTCACAGGTCGTGATATGATTC
AATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGC
GGTAGACAAATTGGATCATTGATTCTCTTTGATTGGACTGAAGGGAGCTCCCTCTctcttttgtattccaattttcttgattaatc
tttcctgcacaaaaacatgcttgatccactaagtgacatatatgctgccttcgtatatatagttctggtaaaattaacattttgggtttatctttatttaagg
catcgccatggataggtgaagaagaatatgaccagttgtggatattcaaatctggtatgacttactttcttcatatctctttagattagcgaatg
2439
>At2g35670 (FIS2)
2440
[Methylated region in the promoter is underlined]
2441
2442
2443
2444
2445
2446
2447
2448
2449
2450
2451
2452
2453
2454
2455
2456
2457
ttatgtagacttattctttggttttactttagtattcaaatttgagatatccttcttcttttttaatctgattttattaaatcattcttaaacttaaaataaaata
aatttctagttgattaccaaacccgaagaagaaaaatttacaataaaacgattttattggacttaaaatgggccatattaacttttaaaattatgaaatt
caacaataattaaggtccaatcgcatatttatttagggtttcgggtatttcatctctataaatatgttatataggtttctcggaaactcagaaatacgccg
caccaacaccaaaaatgaagtttctagctaataagatataatctctctgatactagttttgtgttaaagtggtgaaggcgcaacataaattgttcttcgtt
tgatcattcatttgcgaaatcaaaaacgatcattccgctgcgaaatcataaagtaaatcgaaatctctatagattaaatatgcatgcttagattcagtaa
tattttattaaaatgtaggaatttcaacaatatatatcttgttgtttccatgatttctttttctttttctaattttctattttcttttcttataatctctactgccat
caaaaaattttttaacaaaatttagtctgtttatcaaaattttaatatagatgattcaatattttcatttttaagagcattttgtctgcaaattttaaatgat
atacataatagatttttattaatctcaaaatttggttgtgaagttgtatctacttctgtgatgaagcacacctaatcaaagaagagtcaatctcaaaatat
aagactctttcttacttgtgtcttttcgtagtcatatttttaaaaatgtaaatatattcagcttttatatattcttcaacaaaaaaaaaaatataacaataa
gaagatttcttatttgtagtcttgtagataacgttctcttctattttttcttttaaaaaaaattatgaagttatgtgtagagattctattatgtttcatattggg
aaacatgcacattttccagtccactattctttactctttataatgggtcaaaggtctgccgttacagttttaccttccttactagtttatatatcgcaattgg
gcagctaacattaaatggctatttcatgcacagtgaaagatggttagacgatcattcggagtttcgccaatgatctatacatgtttcattgatttcttgat
atttctgctaattaaaattgcgtggatcacagtctaattcaatgtttatggcgtgacagcttatagattaatcaagcagATGGCTAGGAAGTCCA
TACGGGGGAAGGAAGTGGTAATGGTTTCTGATGATGATGATGATGATGATGATGATGTTGATGATGATAAAAATA
TCATCAAATGTGTCAAACCTCTTACAGTATACAAGAATCTTGAAACTCCAACGGATTCTGATGATAATGATGATGAT
GATGATGATGATGTTGATGTTGATGAAAACATCATCAAATATATCAAACCTGTTGCAGTATACAAGAAACTTGAAA
CTCGCTCAAAAAACAACgtatgcattctcttttttg
2458
>CSTF64 (At1g71800)
2459
2460
2461
2462
2463
2464
Ggtgccatctagacggtcagtaactaataacaacctaatcaataactatacaaatatttttgtccaacatataaaaaaagatattaaaataaatttcct
tttaaccaaaatatcagtttcctttttcggctttcttttttttattcttgttgtagagagagatataaataaatatcgacgacgacggtactaatcgttttgct
aaataatttcattcgtcttctcttcttaactctttaaaaccaaaaactgaaaaataaagacaaaaagtctcctatcgacgacgaatacgaaagatctct
ctcttcagaagaatttaacgaaactcaactgtttcgaattcgatttgaatctcgacccgttacttagcaggaagaagatgaagcagcgcttcaccttctt
cttcgttatcttctcttaataatccccaATGGCTTCATCATCATCCCAACGACGCTGCGTTTTTGgtaactcactcttcttcttcttcttcca
t
2465
>miR159A (At1g73687)
56
2466
2467
2468
2469
2470
2471
2472
2473
2474
cccatgtcttttcagatgcacccacctgttcctttttttcttttctttttattcttctatcacgaatatctaaaccactataattacgactctctatctatcattt
cttccaaaacatgacgtggcctcttctctctctctctttccttctctctaagtatgtttagggttaataattagggttcctcctctcttttgttctgtctttatat
ctccttcatagctctaatgtaagagatttacctcttttggtgtttttgttaatccacgttctcatcaaaactttctcattgttttatgaagagttaaaggtcttt
acagtttgcttatgtcagatccataatatatttgacaagatactttgtttttcgatagatcttgatctgacgatggaagTAGAGCTCCTTAAAGTT
CAAACATGAGTTGAGCAGGGTAAAGAAAAGCTGCTAAGCTATGGATCCCATAAGCCCTAATCCTTGTAAAGTAAA
AAAGGATTTGGTTATATGGATTGCATATCTCAGGAGCTTTAACTTGCCCTTTAATGGCTTTTACTCTTCTTTGGATTG
AAGGGAGCTCTAcatcttctttcaccttctctattttttatttttctttatttctactcaacaattatttattcggattcatctttaattttccgttataattt
ctttttggtaaggattattcgctataatttgagaattatatggttattattttgattccgaagcaatgtttagcgtgttatttgtttttcttgtaaaaaactag
ggtttatgcaactgcctcgggtagatgagaagatccttggttctttggagacttttaaattttgcaatctttgctgccatatgcactgactatctttgtatga
2475
>miR159B (At1g18075)
2476
2477
2478
2479
2480
2481
2482
2483
2484
2485
2486
2487
Aataactagaaaaaagtaagttcttgagtagtaatgaaagtgacaattgctagtaaataagtggtttatgaaatcatatatatgaagtgacaattgct
gtaaatatgtgatttaatagcttttcattaaatatcacaacaaaaaatctgtgaaatgaatcaatagctattaaatcacaaaagtaaaaaaatctgtga
aatgaatcaataatcctaactattaatgggtttaagaaaagtcctcgaaatgaataccatctttgactccctctttctttctcattagggctacagattttt
gtctttaaaaagccattcaagtttcaatggtttttcacttttgttctcctcctccctttttttcttttcaggattcttcttttctatgttttatctttcataatagat
ctgataattttgatttttcactatatatattatggttaatactagtagctttttcatttcaagttttatccttccattggttctttctgagtcaaattgtctcctg
tttcgaaccatatataagttttcaatggttttgtattaactcaagtattcaacattatgtctctctttttcttgcttggatctctaatgctgttcatattttaaa
gcataggtttaggttagatgcatgtaactgccaattaaaagaaggtcaagagttttttgattgtatgaatatatgagttagtcaaagcagatccacacg
attatatagaaaaacaaaggaagaagaagaggAAGAGCTCCTTGAAGTTCAATGGAGGGTTTAGCAGGGTGAAGTAAAGCT
GCTAAGCTATGGATCCCATAAGCCTTATCAAATTCAATATAATTGATGATAAGGTTTTTTTTATGGATGCCATATCTC
AGGAGCTTTCACTTACCCCTTTAATGGCTTCACTCTTCTTTGGATTGAAGGGAGCTCTTcatctctccatccctatctctctctc
tctctatctttagttcttttaagaatgacttttcattttttgtgttgtctgttgcaggtgtataaacatggatgatggatcttgagtaggattttttattttattt
tcttgtgacatatatgcaatttttatggtttgtactttgtatttccattagtttcgtacttttcagttatgtatcattgttaattctaatgtttgaagac
2488
2489
List of oligonucleotides used in this study:
Number
1
Oligo name
GUS-intron-5
GUS-intron-3
3
4
5
6
7
gRNA-F
gRNA-R
pGExp-F1
pGExp-R1
pYPQ131-F
pYPQ131-R
pYPQ132-F
Sequence
aaagcgcgttacaagaaagccgggGTAAGT
TTATCAGTTAAATATAATAA
gttaaaactgcctggcacagcaattgCTGCAT
AAAATCATAAGACCATCG
AGAAATCTCAAAATTCCGGCAGA
GCTGATCCTAAATGCTATCAAG
gtaaaacgacggccagtctta
gcacagcaattgctgcataaaat
ccGGTCTCgctatAGAAATCTCAAAATT
CCGGCAGA
ccGGTCTCccatgGCTGATCCTAAATGC
TATCAAG
ccGGTCTCccatgAGAAATCTCAAAATT
CCGGCAGA
57
Purpose
Amplify an intron to make GUS-intron
construct
Amplify an intron to make GUS-intron
construct
Amplify AtU6 expression cassette
Amplify AtU6 expression cassette
Amplify pGExpress1-gBlock
Amplify pGExpress1-gBlock
Amplify AtU6 expression cassette for
making Golden Gate entry clones
10
pYPQ132-R
11
pYPQ133-F
12
pYPQ133-R
13
pYPQ134-F
14
pYPQ134-R
15
pYPQ135-F
16
pYPQ135-R
17
pYPQ136-F
18
pYPQ136-R
19
pYPQ137-F
20
pYPQ137-R
21
pYPQ138-F
22
pYPQ138-R
23
pYPQ142-F
24
pYPQ142-R
25
pYPQ143-R
26
pYPQ144-R
27
pYPQ145-R
28
pYPQ146-R
29
pYPQ147-R
30
pYPQ148-R
31
pYPQ131B-F
32
pYPQ131B-R
33
pYPQ132B-F
ccGGTCTCggtccGCTGATCCTAAATGC Amplify AtU6 expression cassette for

TATCAAG
ccGGTCTCaggacAGAAATCTCAAAAT Amplify AtU6 expression cassette for
TCCGGCAGA
ccGGTCTCcctggGCTGATCCTAAATGC Amplify AtU6 expression cassette for
TATCAAG
ccGGTCTCgccagAGAAATCTCAAAAT Amplify AtU6 expression cassette for
TCCGGCAGA
ccGGTCTCcaacaGCTGATCCTAAATG Amplify AtU6 expression cassette for
CTATCAAG
ccGGTCTCctgttAGAAATCTCAAAATT Amplify AtU6 expression cassette for
CCGGCAGA
ccGGTCTCctgcaGCTGATCCTAAATGC Amplify AtU6 expression cassette for
TATCAAG
ccGGTCTCgtgcaAGAAATCTCAAAATT Amplify AtU6 expression cassette for
CCGGCAGA
ccGGTCTCcaccgGCTGATCCTAAATG Amplify AtU6 expression cassette for
CTATCAAG
ccGGTCTCacggtAGAAATCTCAAAATT Amplify AtU6 expression cassette for
CCGGCAGA
ccGGTCTCgtttcGCTGATCCTAAATGC Amplify AtU6 expression cassette for
TATCAAG
ccGGTCTCcgaaaAGAAATCTCAAAAT Amplify AtU6 expression cassette for
TCCGGCAGA
ccGGTCTCttcgaGCTGATCCTAAATGC Amplify AtU6 expression cassette for
TATCAAG
GCCTggatccCCAAGTGGTGGCTATCG Amplify AtU6 expression cassette for
AGACC
ggACTAGTgtccAGAGACCACCGGTGG Amplify AtU6 expression cassette for
AAAGCG
ggACTAGTctggAGAGACCACCGGTG
GAAAGCG
ggACTAGTaacaAGAGACCACCGGTG Amplify AtU6 expression cassette for
GAAAGCG
ggACTAGTtgcaAGAGACCACCGGTG
GAAAGCG
ggACTAGTaccgAGAGACCACCGGTG Amplify AtU6 expression cassette for
GAAAGCG
ggACTAGTtttcAGAGACCACCGGTGG Amplify AtU6 expression cassette for
AAAGCG
ggACTAGTtcgaAGAGACCACCGGTG
GAAAGCG
ccGGTCTCgctatTTTACTTTAAATTTTT Amplify AtU3-guide RNA for making
CTTATGG
Golden Gate entry clone pYPQ131B
ccGGTCTCccatgCACAATGTTTTAATT Amplify AtU3-guide RNA for making
TAGGCTCA
ccGGTCTCccatgTTTACTTTAAATTTTT Amplify AtU3-guide RNA for making
58
34
pYPQ132B-R
35
pYPQ133B-F
36
pYPQ133B-R
37
pYPQ131C-F
38
pYPQ131C/DR
pYPQ132C-F
39
40
41
42
pYPQ132C/DR
pYPQ133C-F
43
pYPQ133C/DR
pYPQ131D-F
44
pYPQ132D-F
45
pYPQ133D-F
46
VP64-F (SalI)
47
VP64-R (AatII)
48
NiFls2-gR1top
NiFls2-gR1bottom
NiFls2-gR2top
NiFls2-gR2bottom
NiFls2-gR3top
NiFls2-gR3bottom
NiFls2-gR4top
NiFls2-gR4bottom
NiFls2-gR5top
49
50
51
52
53
54
55
56
CTTATGG
ccGGTCTCggtccCACAATGTTTTAATT
TAGGCTCA
ccGGTCTCaggacTTTACTTTAAATTTT
TCTTATGG
ccGGTCTCcctggCACAATGTTTTAATT
TAGGCTCA
ccGGTCTCgctatGGTACCGAGCTCGG
ATCCACTAG
ccGGTCTCccatgAAGCTTATCGATACC
cTCGACCT
ccGGTCTCccatgGGTACCGAGCTCGG
ATCCACTAG
ccGGTCTCggtccAAGCTTATCGATACC
cTCGACCT
ccGGTCTCaggacGGTACCGAGCTCGG
ATCCACTAG
ccGGTCTCcctggAAGCTTATCGATACC
cTCGACCT
ccGGTCTCgctatAAGGGATCTTTAAAC
ATACGAAC
ccGGTCTCccatgAAGGGATCTTTAAA
CATACGAAC
ccGGTCTCaggacAAGGGATCTTTAAA
CATACGAAC
CGCCGTCGACgatgctcttgacgattttgac
ct
gcgggacgtcCTAaagcatgtcgaggtcgaag
tcat
gattGTTTGAGACGCAGCTCAAAG
Anneal to make the gRNA
aaacCTTTGAGCTGCGTCTCAAAC
gattGGCAAATTACCGTCTGATAT
aaacATATCAGACGGTAATTTGCC
gattGGACAACCTATTTGAGTGGA
aaacTCCACTCAAATAGGTTGTCC
GATTGTTGATGACATGGTTTGAAGA
AAACTCTTCAAACCATGTCATCAAC
GATTGTGCACTTGCATATACTCTC
59

Amplify AtU3-guide RNA for making
Amplify OsU6 guide RNA for making
Golden Gate entry clone pYPQ131C
Golden Gate entry clone pYPQ131C/D
Golden Gate entry clone pYPQ131D
Amplify VP64 (132 bp) from YPQ1 gBlock
Amplify VP64 (132 bp) from YPQ1 gBlock
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
NiFls2-gR5bottom
NiFls2-gR6top
NiFls2-gR6bottom
NiFls2-gR7
(U3)-top
NiFls2-gR7
(U3)-bot
NiFls2-gR9
(U3)-top
NiFls2-gR9
(U3)-bot
NiBAK1-gR1top
NiBAK1-gR1bottom
NiBAK1-gR2top
NiBAK1-gR2bottom
NiBAK1-gR3top
NiBAK1-gR3bottom
NiBAK1-gR4top
NiBAK1-gR4bottom
NiBAK1-gR5top
NiBAK1-gR5bottom
NiBAK1-gR6top
NiBAK1-gR6bottom
FIS2-gRNA1top
FIS2-gRNA1bottom
FIS2-gRNA2top
FIS2-gRNA2bottom
FIS2-gRNA3-
AAACGAGAGTATATGCAAGTGCAC
GATTGACATTGAAGATCCCAACT
AAACAGTTGGGATCTTCAATGTC
GGTCATGCAAATCACCATTGCAAC
AAACGTTGCAATGGTGATTTGCAT
GGTCATGTATACTAATCACTTCAC
AAACGTGAAGTGATTAGTATACAT
gattGTGCTTCTGACACACCTTAT
aaacATAAGGTGTGTCAGAAGCAC
gattGATCACTTCTTTGATGTTCC
aaacGGAACATCAAAGAAGTGATC
gattGAAGCGTATTGCACTTGGGT
aaacACCCAAGTGCAATACGCTTC
GATTGCGAAAAGGAACCATTGAC
AAACGTCAATGGTTCCTTTTCGC
GATTgCACCAAGCAAGAAAAATTGC
AAACGCAATTTTTCTTGCTTGGTGc
GATTGTCAAGTGGGGGCTCCGATTC
AAACGAATCGGAGCCCCCACTTGAC

GATTgTTTACTTTATGATTTCGCAG
AAACCTGCGAAATCATAAAGTAAAc
GATTGTTTTGTGTTAAAGTGGTGA
AAACTCACCACTTTAACACAAAAC
GATTGTCCAATCGCATATTTATTT
60
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
top
FIS2-gRNA3bottom
miR319-gR1top
miR319-gR1bottom
miR319-gR2top
miR319-gR2bottom
miR319-gR3top
miR319-gR3bottom
PAP1-gR1-top
PAP1-gR1bottom
PAP1-gR2-top
PAP1-gR2bottom
PAP1-gR3-top
PAP1-gR3bottom
CSTF64-gR1top
CSTF64-gR1bottom
CSTF64-gR2top
CSTF64-gR2bottom
CSTF64-gR3top
CSTF64-gR3bottom
miR159A-gR2top
miR159A-gR2bottom
miR159B-gR1top
miR159B-gR1bottom
miR159B-gR2top

AAACAAATAAATATGCGATTGGAC
GATTgattgatgaaaaactagaga
AAACtctctagtttttcatcaatc
GATTgttatagatccatgcaaata
AAACtatttgcatggatctataac
GATTgctgttttgaattgatgtttt
AAACaaaacatcaattcaaaacagc
GATTgacagctaatacataaaatg
AAACcattttatgtattagctgtc

GATTgaaaatttataaaaacacgtga
AAACtcacgtgtttttataaattttc

GATTggtataagtaaattggtca
AAACtgaccaatttacttatacc

gattgTTAGTTACTGACCGTCTAGA
aaacTCTAGACGGTCAGTAACTAAc
gattGAAAAAGGAAACTGATATTT
aaacAAATATCAGTTTCCTTTTTC
gattgTAAATAAATATCGACGACGA
aaacTCGTCGTCGATATTTATTTAc
GATTgatagatcttgatctgacga
AAACtcgtcagatcaagatctatc
GATTgaaccatatataagttttcaa
AAACttgaaaacttatatatggttc
GATTgtaactgccaattaaaaga
61
105
AAACtcttttaattggcagttac
gtgtGCGCGCCACCTCGGCCGAAG
aaacCTTCGGCCGAGGTGGCGCGC
gtgtgCCGCGACGAGCACCTTCATG
aaacCATGAAGGTGCTCGTCGCGGc
gtgtGCGGAGAACGACAGCCGGTC
aaacGACCGGCTGTCGTTCTCCGC
tggcaGCGCGCCACCTCGGCCGAAG
aaacCTTCGGCCGAGGTGGCGCGCt
tggcaCCGCGACGAGCACCTTCATG
aaacCATGAAGGTGCTCGTCGCGGt
tggcaGCGGAGAACGACAGCCGGTC
aaacGACCGGCTGTCGTTCTCCGCt
118
miR159B-gR2bottom
YSA-gR1OsU6-F
YSA-gR1OsU6-R
YSA-gR2OsU6-F
YSA-gR2OsU6-R
ROC5gR1OsU6-F
ROC5-gR1OsU6-R
YSA-gR1OsU3-F
YSA-gR1OsU3-R
YSA-gR2OsU3-F
YSA-gR2OsU3-R
ROC5-gR1OsU3-F
ROC5-gR1OsU3-R
NiFLS2-F1
TGCATTAGCCATATTTTCCTTTAC
119
NiFLS2-R2
AATTTCCAAGCTCAGTAGGTATGC
120
NiFLS2-R4
CAGTTTTGTTATAGTTGATGG
121
NiBAK1-F1
TGTCCGTGTTGGTCTACTCTTTTG
122
NiBAK1-R3
ACAACTGCTTCAAAATCCTCATCC
123
YSA-F
ACTTCCTCCCTCTCCCTGT
124
YSA-R
CGCCTTCATCAGTGTGTTG
125
126
127
ROC5-F
ROC5-R
M13-F
CTTTGGGGGCCTCTTTGAC
ATCTGCGTGCGGCGATTC
cccagtcacgacgttgtaaaacg
128
M13-R
ggaaacagctatgaccatg
Amplify deletions mediated by gR1 and

gR2 or gR1 and gR3 or gR4 and gR6 or
gR7 and gR9
gR2
gR3 or gR4 and gR6 or gR7 and gR9
gR3 or gR4 and gR6
gR3 or gR4 and gR6
Amplify for deletions or NHEJ analysis at
OsYSA
Amplify for deletions or NHEJ analysis at
OsYSA
Amplify for NHEJ analysis at OsROC5
Amplify for NHEJ analysis at OsROC5
For sequencing pcr2.1 and nuclease entry
clones
For sequencing pcr2.1 and nuclease entry
clones
106
107
108
109
110
111
112
113
114
115
116
117
62
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
NB-693
NB-694
NB-695
NB-696
NB-697
NB-698
NB-699
AteCas9-seq1
AteCas9-seq2
AteCas9-seq3
AteCas9-seq4
AteCas9-seq5
AteCas9-seq6
hCas9-seq1
hCas9-seq2
hCas9-seq3
hCas9-seq4
hCas9-seq5
hCas9-seq6
Act2-F
Act2-R
PAP1-F
PAP1-R
CSTF-64-F1
CSTF-64-R1
miR319-F
miR319-R
163
linker1-R
164
linker2-F
165
166
linker2-R
linker6-F
FIS2-F1
FIS2-R1
miR519A-F
miR519A-R
miR519B-F
miR519B-R
Linker1-F
aggatgatgatgataagatggc
tctcgtagacttgagaacctta
tagaggaaactctcgtttcgc
actcaagacctacgctcacct
tctgataacgttccatctgagg
aggattctctaaggagtctatc
caaccttggtgctccagctgc
ggctcatctacctcgctctcg
tcagtctaagaacggatacg
tgtcaggtgagcaaaagaa
gtgaaggttgtggatgagttggtg
gttatcaccctcaagtcaaag
aggttgagaagggaaagagtaaga
taccccaccatctaccacctga
tcaagagatacgacgagcaccac
ctgcccaacgagaaggtg
catccagaaagcccaggtgt
gaaagttcgacaatctgacc
caagtgaatatcgtgaaaaagacc
GACCTTTAACTCTCCCGCTATG
AAACCCTCGTAGATTGGCAC
AGTATGGAGAAGGCAAATGGC
CACCTATTCCCTAGAAGCCTATG
CTATCCAGCCATCTCAAGTACC
GACCCTCGTTTATTCTTTTGCC
GAGAGCTTCCTTGAGTCCATTC
TCCCTTCAGTCCAATCAAAGAG
For Sequencing pcoCas9

For sequencing AteCas9
For sequencing hSpCas9
For qRT-PCR analysis
GAGGATAGCGAGAATGAGACTG
CAAACTCTTCACATGCCCAAG
ACATGAGTTGAGCAGGGTAAAG
GGCAAGTTAAAGCTCCTGAGATA
GGGTTTAGCAGGGTGAAGTAA
AAGTGAAAGCTCCTGAGATATGG
gaagGgagacggctctggatcggggtcgggttc Add a 5X GS linker to pNJB185 (pcotggctcaGTCGACgctagcGACGT
dCas9)
CgctagcGTCGACtgagccagaacccgaccc
cgatccagagccgtctcC
catgggagctctctagagctagcgtttaaacacc
ggtgacgt
caccggtgtttaaacgctagctctagagagctcc
catgACCGGTtgggagctcCAATTGTGAT
AAacgt
63
Add a 5X GS linker to pNJB185 (pcodCas9)

linker to replace pco-dCas9 in pNJB185
linker to replace pco-dCas9 in pNJB185
Modify pNJB185 to make pYPQ158 and
pYPQ159
167
linker6-R
TTATCACAATTGgagctcccaACCGGT
168
VP64-F (SalI)
169
VP64-R (AatII)
170
171
174
SRDX3-F (SalI)
SRDX3-R
(AatII)
pYPQ155-F
(D10A)
pYPQ155-R
(D10A)
pcoCas9-F1
CGCCGTCGACgatgctcttgacgattttgac
ct
gcgggacgtcCTAaagcatgtcgaggtcgaag
tcat
CGCCGTCGACcttgatcttgacctcgaactc
gcgggacgtcCTAagcgaatccgagtctaagc
175
pcoCas9-R1
176
pcoCas9-F2
177
pcoCas9-R2
176
pcoCas9-F3
177
pcoCas9-R3
172
173
taagaagtactctatcggactcgCtatcggaact
aactctgtgggatggg
atcccacagagttagttccgataGcgagtccga
tagagtacttcttatcc
CTCCGAATTCGCCCTTCACCATGGAT
TACAAGGATGATGATG
AAATGTTTGAACGATCGGACGTCTCA
CTTCTTCTTCTTAGCCTG
TATCGGACTTGCCATCGGAACCAACT
CTGTTGGATGGGC
TGGTTCCGATGGCAAGTCCGATAGA
GTACTTCT
ATGTTGATGCCATCGTTCCACAGTCT
TTCTTGAAG
GTGGAACGATGGCATCAACATCGTA
ATCAGAAAGT
178
Cas9p-F
aaacaaacgaatctcaagcaatca
Cas9p-R
Cas9p-seq-F1
Cas9p -seq-F2
Cas9p -seq-F3
Cas9p -seq-F4
Cas9p -seq-F5
Cas9p -seq-F6
gactggtgatttttgcggactct
gaagcttgttgactctactga
ccccgagaagtacaaggaga
gcagaagaaggctatcgttg
ttgtcaaggttatgggtcgtc
caccaagtacgacgagaacgacaa
actcccctactgtcgcctact
179
180
181
182
183
184
185
Modify pNJB185 to make pYPQ158 and

pYPQ159
amplify VP64 (132 bp) from YPQ1 gblock
amplify VP64 (132 bp) from YPQ1 gblock
amplify 3X(SRDX) from YPQ2 gblock
amplify 3X(SRDX) from YPQ2 gblock
Introduce D10A mutation based on
pYPQ154 to make pYPQ155
Introduce D10A mutation based on
pYPQ154 to make pYPQ155
Amplify pcoCas9 for making pYPQ150,
pYPQ151 and pNJB185
Amplify pcoCas9 for making pYPQ150,
pYPQ151 and pNJB185
Amplify pcoCas9 for making pYPQ151
and pNJB185
Amplify pcoCas9 for making pYPQ151
and pNJB185
Amplify pcoCas9 for making pNJB185
Amplify pcoCas9 for making pNJB185
Amplify Cas9p from pYLCRISPR-Cas9P35sH plasmid for making pYPQ167
Amplify Cas9p from pYLCRISPR-Cas9P35sH plasmid for making pYPQ167
For sequencing Cas9p
2490
2491
Synthesized gBlock (IDT) DNA sequences in this study
2492
>AtU6-gBlock
2493
2494
2495
2496
2497
2498
ccccatggcgttccctctagataacgcaggatcctaAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAA
ACcAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAA
AATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTC
TAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTA
TATATGGTTAGAcACGAAGTAGTGATTGgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAG
TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGGCAAAAATTTTCAGA
64
2499
2500
2501
2502
2503
TTTTTTCTTCATCTGTAGATTTCTGGGTTTTTTTTTCCGTTTCGTGAATCATAAGTGAAGTTTTGGATGCAAATCTGC
GCGAAAAAAGTTGGACCTGCAATGAGCTTATTTAGATAGCTAAGACAAAGTGATTGGTCCGTTGTTTCAGTTCTGA
TTGTCAGAGAGTTTGTTTCGAGtCGGCGACACCAATGCGTTTTGTTAACCAGATTTCGGGTAAGAAATGTATCGAG
AGTTTGTTTCcAGACGGCTACATCATTTTCTTATGAAGGGTGAAATTAGATAGACCAAAGATTGAAACACAACATTT
CTTTCACAAAAATATAATAAACTTGATAGCATTTAGGATCAGCaactagtaagggcgaattcgaccca
2504
>AtU3-gBlock
2505
2506
2507
2508
2509
2510
2511
2512
2513
ccccatggcgttccctctagataacgcaggatcctaTTTACTTTAAATTTTTCTTATGGCTCAGCCTGTGATGGATAACTGAATCA
AACAAATGGCGTCTGGGTTTAAGAAcATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTT
CCGTTTATATACGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAT
ATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTA
AACAAATCTTGTTGTATATATAACACTGAGGGAGCACCATTGGTCAgagacgagatctagtctgagtcgaccgtctctGTTTTA
GAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
TTCCCTTTCCTTTTTTCTTTTTTTTGCCATAAACTTAAATTTGTATATCGATCATTGTAGATATTGAAAACCTAGAACA
AACCAACATCCATGTGAATGTCTTTCATGACTGATTTAGAGATAATTCTTGAATTTTGGAACTAGAATCTATAATGA
GCCTaaattaaaacattgtgaactagtaagggcgaattcgaccca
2514
>OsU6-gBlock
2515
2516
2517
2518
2519
2520
2521
GGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTGGATCATGAACCAACGGCCTG
GCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCA
TGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGG
GAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTG
TGgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA
CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGCAATTCTGCAGATATCCATCACACTGGC
GGCCGCTCGAGGTCGAgGGTATCGATAAGCTTgaattcgacc
2522
>OsU3-gBlock
2523
2524
2525
2526
2527
2528
2529
2530
ccccatggcgttccctctagataacgcaggatcctaAAGGGATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCA
ACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAGGACAGGC
GTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGGAGT
AAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCG
GCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTT
TAAAAGAGTTGTGCAGATGATCCGTGGCAgagacgagatctagtctgagtcgaccgtctctGTTTTAGAGCTAGAAATAGCAA
GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTCCCTTCGAAGGGC
AATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGGTCGAgGGTATCGATAAGCTTgaattcgacc
2531
2532
>pGExpress-gBlock (synthetic promoter with three miR319 gRNA sites and a mini-35S promoter
highlighted)
2533
2534
2535
2536
2537
2538
2539
gtaaaacgacggccagtcttaagctcgggccccaaataatgattttattttgactgatagtgacctgttcgttgcaacaaattgatgagcaatgctttttt
ataatgccaactttgtacaaaaaagcaggctccgaattcgGGATCCAAGCTTcccatctctagtttttcatcaatcttataaaacgcgcgtaaaaa
ggaagggtccaaaacatgacgtggcctcttctctccacagacagctaatacataaaatgtggatctagttatagatccatgcaaatatggttcagaac
catatataagttttcaatggtccctctcatcgtaaaagcttttctcctttccgtcacgtgtttttataaattttctcaatcagctgttttgaattgatgttttag
gattagatagatcttgatctgacgatggacccaacttctcaaattccaccatttagtaactgccaattaaaagaaggttccttgaccaatttacttatacc
ttaGACGTCAGATCTACTAGTcccttgcaagacccttcctctatataaggaagttcatttcatttggagaggaATGTTACGTCCTGTAGA
AACCCCAACCCGTGAAATCAAAAAACTCGACGGCCTGTGGGCATTCAGTCTGGATCGCGAAAACTGTGGAATTGA
65
2540
2541
TCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGGTAAGTTTATCAGTTAAATATAATAAATAAAGAAGAA
AACCAAAAAAATGGCTAACTAAAACGATGGTCTTATGATTTTATGCAGCAATTGCTGTGC
2542
>gBlock_YPQ1 (containing VP64 sequence)
2543
2544
2545
2546
2547
2548
ttcaccatgggcgaagcttggcttggtaccgatatggactacaaagacgatgacgataaagctcctaagaaaaagcgcaaagtcccctctagataac
gcaggatccgtaagtttatcagttaaatataataaataaagaagaaaaccaaaaaaatggctaactaaaacgatggtcttatgattttatgcagccaa
aaaagaaacgtaaggttGATGCTCTTGACGATTTTGACCTCGATATGCTCGACGCTCTTGATGATTTTGATCTCGACATG
CTCGATGCACTTGATGACTTTGACCTTGACATGCTCGACGCACTCGATGACTTCGACCTCGACATGCTTgagggcaga
ggaagtcttctaacatgcggtgacgtggaggagaatcccggccctatcgattacccttatgatgtgccagactacgctcctaaaaagaagcgcaaagt
tggcattcacggggtgccggctagctaataggcttagatctcttcc
2549
>gBlock_YPQ2 (containing 3X(SRDX) sequence)
2550
2551
2552
2553
2554
2555
ttcaccatgggcgaagcttggcttggtaccgatatggactacaaagacgatgacgataaagctcctaagaaaaagcgcaaagtcccctctagataac
gcaggatccgtaagtttatcagttaaatataataaataaagaagaaaaccaaaaaaatggctaactaaaacgatggtcttatgattttatgcagccaa
aaaagaaacgtaaggttCTTGATCTTGACCTCGAACTCAGACTTGGATTTGCTCTCGATCTCGACCTTGAACTTAGACTCG
GATTTGCTCTTGACCTCGATCTTGAGCTTAGACTCGGATTCGCTgagggcagaggaagtcttctaacatgcggtgacgtggagga
gaatcccggccctatcgattacccttatgatgtgccagactacgctcctaaaaagaagcgcaaagttggcattcacggggtgccggctagctaatagg
cttagatctcttcc
2556
References
2557
2558
2559
2560
2561
2562
2563
2564
2565
2566
2567
2568
2569
2570
2571
2572
2573
2574
2575
2576
2577
2578
2579
2580
2581
2582
Baltes, N.J., Gil-Humanes, J., Cermak, T., Atkins, P.A. and Voytas, D.F. (2014) DNA replicons for plant
genome engineering. Plant Cell, 26, 151-163.
Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V.,
Bogdanove, A.J. and Voytas, D.F. (2011) Efficient design and assembly of custom TALEN and
other TAL effector-based constructs for DNA targeting. Nucleic acids research, 39, e82.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A.
and Zhang, F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science, 339,
819-823.
Curtis, M.D. and Grossniklaus, U. (2003) A gateway cloning vector set for high-throughput functional
analysis of genes in planta. Plant Physiol, 133, 462-469.
Fauser, F., Schiml, S. and Puchta, H. (2014) Both CRISPR/Cas-based nucleases and nickases can be used
efficiently for genome engineering in Arabidopsis thaliana. Plant J, 79, 348-359.
Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd and Smith, H.O. (2009)
Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6, 343345.
Hou, Y., Rajagopal, J., Irwin, P.A. and Voytas, D.F. (2010) Retrotransposon vectors for gene delivery in
plants. Mobile DNA, 1, 19.
Lei, Y., Lu, L., Liu, H.Y., Li, S., Xing, F. and Chen, L.L. (2014) CRISPR-P: a web tool for synthetic singleguide RNA design of CRISPR-system in plants. Molecular plant, 7, 1494-1496.
Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M. and Sheen, J. (2013)
Multiplex and homologous recombination-mediated genome editing in Arabidopsis and
Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology, 31, 688-691.
Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y., Xie, Y., Shen, R.,
Chen, S., Wang, Z., Chen, Y., Guo, J., Chen, L., Zhao, X., Dong, Z. and Liu, Y.G. (2015) A robust
CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and
dicot plants. Molecular plant.
66
FLAG NLS
IV2 intron
NLS
pcoCas9
pYPQ150
FLAG NLS
IV2 intron
NLS
pcoCas9
pYPQ151
D10A
FLAG NLS
IV2 intron
pYPQ152
NLS VP64
pcoCas9
D10A
FLAG NLS
H840A
IV2 intron
5X GS linker
NLS 3X(SRDX)
pcoCas9
pYPQ153
D10A
H840A
5X GS linker
NLS
pYPQ154
AteCas9
NLS
pYPQ155
AteCas9
D10A
3X FLAG NLS
pYPQ158
NLS
hSpCas9
3X FLAG NLS
pYPQ159
NLS
hSpCas9
D10A
NLS
NLS
pYPQ167
Cas9p
Figure S1. Architecture of Cas9 proteins in the toolbox

Architecture of different Cas9 proteins is depicted. Note these Cas9 proteins differ from each other
based on different epitope tag or effector protein fusions.
A
BsaI
CCAG gR4 TGTT
BsaI
TGTT gR5 TGCA

tet
pYPQ135
tet
pYPQ134
BsaI
GGAC gR3 CCAG
BsaI
BsaI
pYPQ133
CATG gR2 GGAC
BsaI
BsaI
pYPQ132
CTAT gR1 CATG
tet
BsaI
tet
BsaI
BsaI
pYPQ131
GAAA gR8 TCGA
pYPQ138
tet
attL5 CTAT
CGGT gR7 GAAA
BsaI
pYPQ137
tet
BsaI
TGCA gR6 CGGT
pYPQ136
tet
BsaI
BsaI
BsaI
LacZ
BsaI
BsaI
tet
TCGA attL2
pYPQ148
spec
attL5 gR1 gR2 gR3 gR4 gR5 gR6 gR7 gR8
attL2
pYPQ148-gR1-8
spec
3kb
1kb
Digested by BamH1 and EcoRV
Vector backbone
Figure S2. Golden Gate assembly of up to 8 gRNA expression cassettes

A, A depiction of Golden Gate assembly of 8 gRNA expression cassettes into one recipient vector
based on compatible ends resulting from BsaI digestion. B, Restriction digestion of plasmids derived
from randomly selected white colonies (on blue/white selection LB plate) to confirm Golden Gate
assembled vectors. Note the successfully assembled vectors that contain 2, 3, 5 and 8 gRNA
expression cassettes are indicated by red stars.
B
NbFLS2
NiFLS2
NiFLS2
NbBAK1
D
1
2 1 2
1 2 (-)
(-)
(-)
2 (-)
WT
* * * * * *
* *
* *
* *
Deletions at NbFLS2
G
H
Deletions at NbFLS2
Deletions at NbFLS2
NbFLS2-deletion by gR1 and gR2

GTTTGAGACGCAGCTCAAAGGGG//GGCAAATTACCGTCTGATATTGG
GTTTGAGACGCAGCTCA---------------------------TATTGG
GTTTGAGACGCAGCTCA----------------------------ATTGG
GTTTGAGACGCA----------------------------------TTGG
GTTTGAGACGC----------------------------------ATTGG
Deletions at NbBAK1
(genomic)
(-307 bp, 9X)
(-308 bp)
(-314 bp)
(-314 bp)
NbFLS2-large deletion by gR1, gR2 and gR3

GTTTGAGACGCAGCTCAAAGGGG//CCCTCCACTCAAATAGGTTGTCC (genomic)
GTTTGAGACGCAGCTCAA----------------CTCAAATAGGTTGTCC (-777 bp)
GTTTGAGACGCAGCTCA-----------------CTCAAATAGGTTGTCC (-778 bp)
NbFLS2-large deletion by gR4, gR5 and gR6
CCTTCTTCAAACCATGTCATCAAC//CCGAGTTGGGATCTTCAATGTC (genomic)
CCTTC----------------------------TTGGGATCTTCAATGTC (-784 bp)
CCTTC------------------------------GGGATCTTCAATGTC (-786 bp)
J
K
L
NbFLS2-large deletion by gR7 and gR9

ATGCAAATCACCATTGCAACTGG//ATGTATACTAATCACTTCACTGG (genomic)
ATGCAAATCACCATTG----------------------------CACTGG (-647 bp)
ATGCAAATCACCAT------------------------------CACTGG (-649 bp)
NbBAK1-large deletion by gR1, gR2 and gR3
CCTATAAGGTGTGTCAGAAGCAC//GAAGCGTATTGCACTTGGGTCGG (genomic)
CCTATAA--------------------------------------GTCGG (-2055 bp)
CCTATA--------------------------------------GGTCGG (-2055 bp)
NbBAK1-large deletion by gR4, gR5 and gR6
CCAGTCAATGGTTCCTTTTCGC//CCTGAATCGGAGCCCCCACTTGAC (genomic)
CCAGTC--------------------------TCGGAGCCCCCACTTGAC (-2039 bp, 2X)
Figure S3. Targeted chromosomal deletions at NbFLS2 and NbBAK1 in N. benthamiana

Schematics in (A) and (B) depict target genes and relative positions of gRNA binding sites in NbFLS2
and NbBAK1 respectively. C-E, PCR based detection of deletions at NbFLS2. The deletion bands are
indicated by asterisks. Note the larger PCR products are corresponding to wild type (WT) sequence.
The numbers indicate biological replicates and the wild type control plant is labeled as (-). F, PCR
based detection of deletion at NbBAK1, with the deletion bands indicated by asterisks. G-L, Sequence
confirmation of different lengths of deletions at NbFLS2 and NbBAK1 induced by different gRNA pairs.

A CRISPR-Cas9 Toolbox For Multiplexed Plant Genome Editing and Transcriptional Regulation - 2015

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A CRISPR-Cas9 Toolbox For Multiplexed Plant Genome Editing and Transcriptional Regulation - 2015

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Breakthrough Technologies

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome

Customizable sequence-specic nucleases (SSNs)

homologous recombination (Kanaar et al., 1998; Puchta,

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

surfaced, a more versatile transcription activator-like

nickases (Gasiunas et al., 2012; Jinek et al., 2012). While

We sought to design a multifaceted and easy-to-use

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

destination vectors have been previously developed

Plant Physiol. Vol. 169, 2015

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

three copies of the transcriptional repressor domain

unique applications and capabilities of our CRISPR/

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

Table I. Vectors in the plant multiplex CRISPR/Cas9 toolbox

Golden Gate entry vector; first gRNA under AtU6 promoter

assembly. This strategy of cloning relies on Type IIS

using BsaI as our Type IIS restriction endonuclease

Plant Physiol. Vol. 169, 2015

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

and OsU3 promoters, we generated Golden Gate

Targeted Chromosomal Deletions in Tobacco

We rst tested our system for creating targeted

CRISPR/Cas9 system allows effective expression of

Targeted Deletion and Simultaneous Mutagenesis in Rice

Having shown our multiplex Cas9 system works

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A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

mutations are predominantly biallelic (Supplemental

Plant Physiol. Vol. 169, 2015

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Figure 4. Activation of coding and noncoding

Targeted Transcriptional Activation of Protein-Coding and

A primary design goal for our vector toolbox was to

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

Borevitz et al., 2000) and miR319 (a microRNA; Weigel

cytosine sites include CpG, CpHpG, and CpHpH (Law

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rosette leaves (Fig. 5C). All analyzed lines showed

Our toolbox also enables transcriptional repression

the control (Fig. 6D). Similarly, all three independent

A critical aspect of designing multiplex CRISPR/Cas9

Figure 6. Multiplex and simultaneous gene repression in Arabidopsis by dCas9-3X(SRDX). A to

Plant Physiol. Vol. 169, 2015

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A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

very well in our system. Although we have focused on

Plant Physiol. Vol. 169, 2015

Downloaded from www.plantphysiol.org on December 21, 2015 - Published by www.plant.org

In this study, we developed and tested a multifaceted

Transient Expression in Tobacco

Arabidopsis Transformation and Screen

Rice Protoplast Isolation and Transformation

A. tumefaciens-Mediated Transformation of Rice

Assays for CRISPR/Cas9 Activity in Vivo

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A CRISPR/Cas9 Toolbox for Gene Editing and Transcriptional Regulation

RNA Extraction and cDNA Synthesis

Quantitative PCR and Data Analysis