The CRISPR-Cas System: Background, Significance, and Potential

TopicsinPharmaceuticalSciences
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TheCRISPR-CasSystem:Background,Significance,andPotential
DavidWych

Introduction
CRISPR (short for Clustered Regularly Interspaced Short Palindromic Repeats)
is the name given to small,patternedsegmentsofprokaryoticDNAthat,togetherwith
CRISPR-associated (Cas) protein complexes, and associated RNA tags, allow for the
generation of highly-customizable, highly-efficient, targeted nucleases (DNA/RNA
slicers). This system was discovered by accident in the late 1980s, and was later
demonstrated to function as a quasi-immune system for prokaryotic organisms:
short segments of viral DNA from past attacks are stored between recognizable
palindromic nucleotide repeat markers in the genome; once transcribed, associated
enzymes use the complementary RNA-segments as tags to recognize and cleave the
invader DNA when the next attack occurs. These snippets of viral DNA and the
associated Cas family of proteins act as a dynamic, inheritable defense mechanism
against phages and plasmids.Sincetheirinitialdiscovery,theCas9proteinsystemand
associated RNAs (crRNA and tracrRNA), has been found to act as a simple, cheap and
highlyefficientsequence-specificDNAcleavagesysteminallorganismsinwhichithas
been tested to date, and has emerged as themostpromising,powerfulandpotentially
harrowinggeneticediting/engineeringtooleverdiscovered.
This paper will first give a briefhistoryofthediscoveryandinvestigationofthe
CRISPR-Cas system, then present an overview of the system and its applications as
they are understood today, and end with a few current areas of interest and
controversy.

DepartmentofPharmaceuticalSciences-UniversityofCaliforniaIrvine
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DiscoveryandElucidationofFunction
In 1987, Yoshizumi Ishino et al. conducted a gene-sequence and gene-product
analysis experiment to investigate the iap gene locus of E. coli, which was thought to
code for an enzyme that mediates the maturation of isozymes of alkaline
phosphatase[1]. In the course of analysing the sequence and suggesting a likely gene
product, they found an unusual nucleotide structure in the 3 flanking region of the
locus:
Five highly homologous sequences of 29 nucleotides were arranged as direct
repeats with 32 nucleotides as spacing. So far, no sequence homologous to
these has been found elsewhere in procaryotes, and the biological significance
ofthesesequencesisnotknown.
A few years later, Nakata et al. were able to identity many instances of the same 29
base pair repeat with 32/33 base pair spacer sequences downstream of the iap gene,
and elsewhere in the E. coli genome
(Fig. 1), in addition to the genomes of
two other bacterial organisms (in the
Shigella and Salmonella genera)[2]. In
1995, Mojica et al. identified the same
structure in twospeciesofArchaea,and
suggested that it is involved in replicon
(prokaryotic chromosome)
partitioning[3]. Five years later, Mojica
(with different collaborators) published a MicroCorrespondence in the Journal of
Molecular Microbiology highlighting the significance of thisstructure,suggestingthey
maybeessentialtothegenomeofallprokaryotes[4].
The first published usage of the mnemonic CRISPR came from a paper by
Jansen et al in 2002, which also identified four CRISPR-associated (cas) genes located
adjacent to CRISPR loci (many organisms have multiple separate CRISPR gene loci),
and suggested that the two gene loci have a functional relationship[5]. In addition, the

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CRISPR-cas lociwerefoundtobelargelyconservedwithinaspeciesbutnotconserved
between species. The cas genes showed motifs consistent with helicases and
exonucleases, suggesting that these genes are involved in DNA metabolism and gene
expression. Bolotin et al., in 2005, discovered that the spacers between CRISPRs were
of extrachromosomal origin, due to homology with phage gene sequences, and
suggested(presciently):
[T]he spacer elements are the traces of past invasions by extrachromosomal
elements [and] provide the cell immunity against phage infection, and more
generallyforeignDNAexpression,bycodinganantisenseRNA.[7]
Pourcel et al. echoed this suggestion in a paper published in the same volume that
recorded the addition of a new spacer to the CRISPR locus, and confirmed that the
newspacerwastakenfromprophageDNA[6].
The following year, Makarova et al., working out of the National Institutes of
Health, published an exhaustive sequence analysis of the cas gene locus, allowing for
the classification of 6 distinct gene superfamilies therein (cas1-6). In addition, they
made homology arguments hypothesizing the activity of cas-derived proteins, and
gave the first rough sketches of the hypothetical workings of the CRISPR-Cas system
(which they dub CASS) as a defence mechanism against invading phages, and the
enzymatic system for incorporation of new CRISPR units into the locus. They also
demonstrated that new inserts to the locus showed almost no similarity with even
closely related prokaryotic strains, which suggested rapid (non-evolutionary-time)
turnover of the genes[8]. With an eye toward the future, they remarked,itseemsthat,
once the psiRNA [prokaryotic silencing RNA] mechanism described here is
investigated experimentally,itcouldbeexploitedtosilenceanygeneinorganismsthat
encodeCASS.
The first comprehensive, direct experimental demonstration that the
CRISPR-cas locus is responsible for inheritable adaptive phage immunity came in
2007, in a short, landmark paper by Barrangou et al., publishedinScience.Thispaper
definitively showed that CRISPR spacers areresponsiblefortargetedphageresistance

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(both inherited and acquired), and that cas-derived proteins are the enzymatic
machinery behind phage DNA neutralization (N.B. in the early months of 2017, the
paperhasbeencitednearlytwothousandtimes)[9].
It is important to note, though, that at this time, the enzymatic mechanism
responsible for phage resistance was still largely unknown. Research up to this point
was able to demonstrate that the CRISPR-cas system acts as a nucleic acid-based
phage resistance/immunity system, but the real power of the system (the Cas
enzymatic machinery, and its applicability in other organisms) was yet to be
discovered.

InvestigationofEnzymaticMechanisms
In the following years, the nucleic acid-/enzyme-level mechanism behind
CRISPR-Cas-mediated phage immunity was pieced together. Marraffini and
Sontheimer published work demonstrating that Cas-protein interference works by
targeting DNA directly (in contrast with RNA interference, or RNAi, mechanisms that
had been previously observed in
eukaryotic organisms) by showing that
short sequences of RNA transcribed from
the CRISPR locus (dubbed crRNA) were
responsible for the targeting of particular
phage DNA segments[13]. They were also
able to show in a laterpaperthatelements
of thespacerregionflankingtheprocessed
crRNA spacer region are responsible for
self versus non-self recognition, an essential component of any immune system (Fig.
2)[18]. Andersson et al. providedevidenceofthisadaptiveimmunitymechanismatwork
in a Leptospirillum population in the presence of viral plasmids, but also showed that
recombination of viral DNA allowed for evasion of the CRISPR mechanism[11] -- as an

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aside, this work also illustrated that viral immunity mediated by the CRISPR-Cas
system is a dynamic process, shaped by forces at the population level in both the
bacteria andviruses,andthatCRISPR-mediatedphageresistancecanbegainedorlost
in a time scale of months or less. Work by Deveau et al. uncovered asmall,conserved
sequence of nucleotides flanking the proto-spacer (the name given to the segment of
phage DNA complementary to the CRISPR spacer), later termed the
protospacer-adjacent motif (PAM), and determined that, in addition to allowing for
self versus non-self discrimination as shown previously, it also is necessary for
recognition by the Cas enzymatic complex[14] (confirming suggestion put forward of
Horvath[12]etal.fromthesameyear).
In a paper published in Cell in 2009, Hale et al. would provide the first
experimentalisolationanddetailedstudyofaCaseffectorcomplexinP.furriosis.They
were the first to show that the effector complex is comprised of the spacer-derived
RNA (psiRNA) and an attached, conserved 5 spacer-derived psi-tag, together with a
complex of Cas proteins[15]; they also demonstrated that this particular Cas protein
complex splices invader RNA strands complementary to a site 14 base-pairs from the
3 end of the psi-RNA. In a 2010 paper in Nature, Garneau et al. would demonstrate
that this mechanism works on both bacteriophage and plasmid DNA, and that the
mechanism cleaves double-stranded DNA as well[19]. Taken together, these results
suggested that the CRISPR-Cas system could be used as a targeted cleavage
mechanism for any nucleic acid sequence. In 2011, Deltcheva etal.discoveredanother
CRISPR-associated RNA product (trans-activating CRISPR RNA, or tracrRNA, for
short) which is complementary to the repeat region, and is important for the
maturation of the crRNA before it can guide Cas proteins to the target invader
sequence[20].
Sapranauskas et al., in 2011, were thefirsttodemonstratethattheCRISPR3/Cas
gene locus from S. thermophilus could be transferred to E. coli (in a different
phylogenetic kingdom), and would provide protection against plasmid and phages[22].
Theyalsoconfirmedtheresultthattheproto-spaceradjacentmotif(PAM)isnecessary

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for immunity, and demonstrated that Cas9 is the only protein necessary for
CRISPR-mediatedcleavage(inthisparticularCRISPR-Cassystem).
Faced with the growing interest in the CRISPR-Cas system, several teams of
researchers began publishing work intended to classify different variations of the
system, and standardize nomenclature[23]. In 2011, Makarova et al. examined the
CRISPR/Cas system from an evolutionary point of view; this work is responsible for
the delineation of the three main classes of CRISPR systems (types I, II, and III) and
subtypes therein, which differ both in the way new phage DNA spacers are
incorporated into the locus, and the way in which the crRNA is processed by the
various Cas proteins before guiding the effector complex to the target; they also
presented the taxonomic distribution of the three main systems across Archaea and
Bacteria[21]. As an aside, Dr. Kira S. Makarova appears to be the communitys foremost
expertonthegeneticsandevolutionoftheCRISPR-caslocus.
The first team of researchers to demonstrate that the CRIPSR-Cas could be
used as a broadly-applicable efficient, targeted genome editing toolwasJineketel.,in
2012[27]. This paper signalled a major shift in focus, with respect to the interest in the
CRISPR-Cas system from prokaryotic curiosity to powerful tool. However, before the
methodology of Jinek et al. can be appreciated, it will be necessary toprovidearough
sketch of the workings of the system -- from spacer acquisition to
phage-DNA-intervention.

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TheCRISPR-CasMechanism-FromAcquisitiontoInterference
Acquisition
When a CRISPR-endowed prokaryote is challenged by a virus or plasmid, and
foreign DNA/RNA is released into the cytoplasm, Cas1 and Cas2 (the only proteins
conserved across all three types of CRISPR-Cas system[21]) form a complex[54] which
binds to the invading strand, cleaves off a protospacer, and incorporates the new
spacer into the CRISPR locus[24]. The protospacer is not randomly chosen by the
Cas1-Cas2 complex: a 2-5 nucleotide protospacer adjacent motif (PAM) serves as a
recognition signal for the initiation of protospacer cleavage[25]; in type II CRISPR, one
nucleotide from the PAM is inserted as the last nucleotide of the new repeat during
incorporation[44].
During incorporation, the first repeat adjacent to the leader (promoter)
sequence is copied, and the new spacer is inserted between the copies[69]. It is known
that Cas1 is responsible for the enzymatic cleavage of the invader sequence, and that
the Cas1-Cas2 complex is uniquely responsible for recognition of the CRISPR locus
leader sequence and new spaceracquisition[54],however,therearemanyaspectstothe
adaptation phase still unknown. It is also important to note that theacquisitionphase
is not identical across all CRISPR types, though the general model of insertion is
generallyquitesimilar.
crRNABiogenesisandProcessing
When signalled (e.g. by upregulation through increased cytosolicconcentration
of cAMP[18]), the CRISPR-cas locus is transcribed, creating a long precursor RNA
(pre-crRNA) transcript containing strands complementary totheentirerepeat-spacer
sequence.
In Type I and III systems, processing (cleavage) of the pre-crRNA iscarriedout
by Cas6: the pre-crRNA transcript is cut in the repeat sequences, leaving an

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8-nucleotide 5-handle, and a 3-handle that varies in size, and sometimes folds back
ontoitselftoformahairpin(stem-loop)structure[41,70].
Type II systems contain a portion of the repeat sequence that is transcribed to
form what is known as trans-activating RNA (tracrRNA)[20]. Tracr-RNA contains a
sequence that is complementary to a portion of therepeatsequenceofthepre-crRNA
transcript; upon base-pairing with this region, the tracrRNA-pre-crRNA duplex is
bound byCas9,whichrecruitstheRibonucleaseIII(aubiquitousdouble-strandedRNA
processing enzyme), which cleaves the pre-crRNA strand, leaving a mature crRNA
strandcomplexedwithCas9[27].

EffectorComplexFormation
In Type I andIIICRISPRsystems,thematurecrRNAstaysboundtoCas6,which
recruits the other Cas proteins to form a CRISPR-associated Complex for Antiviral
Defense, dubbed Cascade. In Type I systems, the Cascade complex is composed of
one Cse1, two Cse2, one Cas5, six Cas7 and one Cas6 proteins[71]. The overall complex
has a seahorse-shaped architecture,withthesixCas7proteinsformingahelix-shaped
backbone, which binds, stabilizes and displays the crRNA[23,50]. The Type III Cascade
complex, though composed of different proteins, has a similar structure to the TypeI
complex. Indeed, the backbone of the complex is composed of multiple copies of a
protein that binds the crRNA and has been shown to be structurally homologous to
Cas7 (Hrle et al 2013). However, they are not completely homologous. In fact, in

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addition to differences between the Types, the Type I and III systems themselves are
divided into subtypes based on differences in the structure and function of the
respective effector complexes: Types I-A, I-C, I-E, and I-F, as well as Types III-A and
III-B. A diagram of the differences in the composition of the effector complex is
providedinFigure4,alongwithacrystalstructureofaTypeIcomplexinFigure5.

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In Type II systems, the effector complex consists only of the Cas9 protein,
crRNA guide, and tracrRNA. The Cas9 protein has been shown to consist of an
-helical lobe, whichcoordinatestheinteractionofthecrRNA-targetinteraction,and
a nuclease lobe, which recognizes the PAM and cleaves the target DNA[49]. Cas9
stabilizes the crRNA and exposes it for base-pairing with the complementary invader
strand, but the crRNA also acts as an agonist for Cas9:uponbindingofthecrRNA,the
two lobes undergo a conformational change that allows for DNA binding and
cleavage[55].

TargetRecognitionandInterference
In all three CRISPR systems, recognition of the invader DNA/RNA proceeds in
roughly the same manner[33]: the invading nucleotide sequence is non-specifically
bound and scanned; if the PAM sequence is recognized, the target DNA is unwound,
forming an R-loop[23]; there is an initial base-pairing event at a 7-12 nucleotide
sequenceknownastheseedregion,followedbyextendedpairingwiththerestofthe
protospacer; once the entire protospacer is bound, the effector complex undergoes a
conformational change that allows for cleavage of the invader sequence. It is in this
finalstepthatthethreetypesofsystemsdiffer.
In Type I systems, once the seed regionhasbasepairedtothecrRNAguide,the
invader DNA forms an R-loop, which triggers a conformationalchangeintheCascade
complex and encourages the recruitment of Cas3 (Westra et al 2012), which is an
ATP-dependent helicase and a metal-dependent nuclease. Upon binding of Cas3, the
DNA strand is positioned by the Cascade complex for degradation.InTypeIIsystems,
once the seed region has been paired, Cas9 (which has already adopted a
nuclease-ready conformation,thankstobindingwiththecrRNA)usesitsownintrinsic
nuclease activity to cleave the invader DNA sequence. Cas9 has an HNH-like nuclease
domain that cleaves the DNA strand base paired with the crRNA, and a RuvC-like
nuclease domain that cleaves the other strand[27,55]. In contrast with the Type Isystem,
TypeIIDNAdegradationisnotATP-dependent.

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The mechanism by
which Type III systems
target invader
polynucleotides is still
largely unknown, however
there are a few details
known about the process
to date.Forone,TypeIII-A
systems appear to
discriminate self from
nonself DNA in a manner
that is
PAM-independent[13]. Type
III-B systems, remarkably,
are unique among known
CRISPR systems in that
they target RNA instead of
DNA[15] (though ithasrecently
been purported that the differences in activity betweenTypeIII-Aand-Bsystemshas
been resolved, and they are essentially structurally/functionally homologous[68]).
Beyondthis,thereismuchstilltobediscoveredwithregardtoTypeIIIsystems[65].
TheProtospacerAdjacentMotif
Though it has been mentioned already, it is important to note the significance
of the protospacer adjacent motif (PAM). A paper by Sternberg et al. in2014[52]showed
that for the Type II CRISPR system both binding and cutting by Cas9 require PAM
recognition, and that off-target binding affinity scales with the densityofPAMmotifs.
More remarkably, sequences that match the complementary guide-RNA, but lack the

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PAM are avoided by Cas9. They also showed that base-pairing with the target DNA
starts at the PAM, and that PAM interactions trigger Cas9 nuclease activity. In
summary, not only does thePAMallowforselfversusnon-selfdiscrimination(thekey
component of any successful immunesystem),intheTypeIICRISPR-Cas9systemitis
arguably the single most importantfactorintargetrecognitionandbinding,DNA/RNA
heteroduplexformationandnucleaseactivity.
Reviews of the System throughtime:MarraffiniandSondheimer(2010)[17]

,Deveauetal.(2010)[16]
,Gasiunaset
al.(2012)[28]
,vanderOostetal.(2014)[57]
,Sanderetal.(2014)[60]

ApplicationsandLimitations
Armed with knowledge of the details of the enzymatic machinery behind
CRISPR-Cas interference, the landmark work of Jinek et al. can bebetterunderstood.
To begin with, it should be noted that Type II CRISPR systems have a variety of
logistical strengths compared to Type I and III systems, with regard to their use as
enzymatic tools. For one, the Type II system is simpler,
and has been studied in greater detail. More
pragmatically though, Type II systems utilize only Cas9
(instead of large multi-protein effector complexes)
which has intrinsic helicase and nuclease activity (in
contrast to Type I and III systems which require the
recruitment of other Cas proteins). Through the use of
various assays, Jinek et. al were able to prove that
tracrRNA is required for for target DNA recognitionand
that PAM recognition is an important factor in initial
DNA duplex unwinding,crRNAbase-pairing,andR-loop
formation. In addition, and most remarkably, they were
able to show that Cas9 can be programmed using a
single engineered chimeric RNA strand that performs

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the role of both tracrRNA and crRNA. These chimeric-RNA-guided nucleases were
shown to be efficient and reliable, as well as incredibly cheaptomanufacture.Also,in
contrast to existing gene editing tools such as TALENs or Zinc-Finger Nucleases, no
modifications need to be made to the enzymatic protein itself -- allthatisrequiredis
the manufacture of a single RNA strand, and the presence of the (Guanine-Guanine)
PAMinthetargetstrand.TheCRISPRcraze[72]wasabouttobegin.
In subsequent years, various labs were able to use the Type II CRISPR/Cas9
system todemonstratesite-specificchromosomalDNAcleavageandgeneactivationin
the genomes of various plant, insect, and mammal species, including the human
genome.
Multiple labs were quickly able to use CRISPR-Cas systems to efficiently alter
the genome of plants, such as wheat and rice. Kabin XieandYinongYangwereableto
show that the PAM ispresentatafrequencyof
about one PAM for every 10 base-pairs in the
rice genome,suggestingthatmorethan90%of
rice genes are available for targeting by
CRISPR-Cas9[47]. In 2014, Shan et al. were able
to use the CRISPR-Cas9 system to
demonstrate that specific genes in rice
protoplasts could be targeted and mutated
within 1-2 weeks, and mutated rice plants
couldbesuccessfullygrownin13-17weeks[62].
Bin Shen et al. were the first topublish
results confirming that CRISPR-Cas9 could be used to inducesite-specificcleavageof
loci within the mouse genome[45]. In the same year, Li Dali et al. would publish work
confirming these results, and performing the same protocol with rats[48]. This work
would be followed up by a study in 2014 by Haoyi Wang et al. who demonstrated the
use of multiplex signal RNA to simultaneously disrupt fivemouseembryonicstemcell
genes with high efficiency - this allows for the one-step generation of mice with

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tailored mutations to multiple specific genes[61]. This breakthrough has the potential
greatlyacceleratethepaceofdrugdiscoveryandclinicaltesting.
CRISPR-Cas9 would not only revolutionize existing gene-targeting animal
models, but also allow for the creation of new ones. In 2014, Dongshang Yang et al.
were able to use CRISPR-Cas9 to create gene-targeting-transgenic (GTT) rabbits for
the first time, generating 4 novel linesofknockoutrabbits[63].Similarsuccesswasseen
in the targeting of the genomes of various organisms, from silkworms, to frogs and
zebrafish[40].
Multiple labs were also able to demonstrate the use of Cas9 to affect gene
expressioninhumancells.Cong.etal.demonstratedtheuseofboththestandardCas9
system and a modified Cas9 nickingenzymethatonlycleavesonestrandtofacilitate
sequence-homology-directed genomic insertions and repairs in mammalian cells[35,56].
They were also able to demonstrate the use of multiplex genome engineering: usinga
single connected RNA-array to target multiple separate gene loci in series --
essentially demonstratingtheabilitytocreatealgorithmicgene-editingprogramsbuilt
into in a single RNA strand. Mali et al. would echo these results, using the multiplex
CRISPR-Cas9 system to target genes in three types of human cells, including
pluripotent stem cells[38,42]. Though these initial results were exciting, much caution
had been exercised in the use of this technology to alter the human genome, limiting
testing to laboratory cell lines and stem cells. To the surprise of many in the field,
Liang et al., working out of Sun Yat-sen University in Guangzhou, China published
research in 2015 demonstrating theyhadeditedthegenomeofhumanembryosforthe
firsttime[67].
It was soon discovered that the system is not only useful for its DNA-cutting
functionality, but also for its highly-specific targeting functionality alone[30-34]. Qietal.
made a significant breakthrough in 2013 with the creation of the CRISPRi (short for
CRISPR interference) system which utilizes dCas9, a mutated form of Cas9 that lacks
all endonuclease activity[39]. They used this neutered form of Cas9, along with
guide-RNAs, to trigger the activation and repression of gene transcription in human

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cells. This method is fundamentally different than previous methods, which relied on
the generation of sequence-specific DNA binding proteins, and opened the door for
the fast, inexpensive, highly-specific, reversible gene activation and repression tools
with few off-target effects. They also showed that this system could be used to
regulate multiple genes at once -- the small size of the targeting region of the
guide-RNA (20 base-pairs) allowsforamultitudeoftargetsonthescaleof(potentially)
the entire human genome. This work was echoed by Pirez-Pinera et al. who used the
same system to reliably and reversibly activate human genes[29]. Gilbert et al. and
Maeder et al. demonstrated the use of a similarsystem,witheffector-domainsferried
by the dCas9 to particular gene loci, to recruit proteins and cause transcriptional
activationandrepressioninhumancells[30,43].
This lift-off phase was not without signs ofpotentialdanger.Fuetal.published
a paper in Nature Biotechnology demonstrating that ribonucleotide guided nucleases
(RGNs), CRISPR-Cas9 among them, can cut off target sites that differ from the
complementary strand to the guide by up to five nucleotides[36]. Fu would publish
another result the following year (with a different team ofcollaborators)showingthat
the use of truncated guide-RNAs (with less than twenty nucleotides the in the
target-complementary segment) can reduce off-target mutagenesis by 5000-fold or
more without sacrificing accuracy[51]. However, off-target effects are still a serious
concern[58], and (along with difficulties in the delivery of the RNA/Cas-complex to the
nucleus of target cells) constitute one of the most significant roadblocks for the
CRISPR-Cas9 system as a potential drug model. The potential complications were
certainly not a deterrent for applications for intellectual property over the use of the
CRISPR-Cas9system.Quitetheopposite...

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2017PatentDispute
In 2012 Jennifer Doudna, of the University of California, Berkeley filed for a
patent on the intellectual property rights to the CRISPR-Cas9 system, showing that it
can be used to modify specific loci of the bacterial genome. Dr. Doudna, and her
frequentcollaboratorEmmanuelleCharpentier,aregiantsinthefield,andhavebeena
part of many major discoveries (including the seminal Jinek et al. work referenced
above, upon which thepatentwasbased,andtheinventionoftheCRISPRisystem,just
to name a few). Shortly after, Feng Zhang, of the Broad Institute of MIT and Harvard,
was granted a patent for the use of the same CRISPR system to edit the genome of
eukaryotes.
Recognizing the potentially astronomical financial incentive in the future, the
University of Berkley sued to have the Broad Institutes patent thrown out, arguing
that Zhang was awarded a patent over the same technology Doudna and her
collaboratorshadinvented,merelyusedinadifferentcontext.
On February 15th, 2017, the United States Patent and Trademark Office ruledin
favor of Zhang, claiming the Broad Institutes legal representation had successfully
convinced governmentthattheinnovationwasspecifictotheeukaryoticenvironment,
and not merely an obvious re-application of Doudnas invention. However, although
Zhangs patent claim over the use in eukaryotes was upheld, Doudna still retains her
patent over the use of the CRISPR-Cas9 system not restricted to any environment.
Interviewed by the Washington Post about the decision, Doudna commented, They
will have a patent on green tennis balls. We will get a patent on all tennis balls.
Whether or not the University of California will appeal the ruling is yet to be seen.
Considering theprecedentsetbytheupholdingofZhangspatent,itwillbeinteresting
to see how future patent battles play out, if other labs attempt to patent equally
specific implementations of CRISPR-Cas technology. Given the multitude of
differences between Types and Subtypes of CRISPR I, II and III systems, the potential
fornewinnovationsspecifictovarioussystems/organismsseemshigh.

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EthicalConcerns
It is tempting to underestimate the potential of the CRISPR-Cas system. In
many ways, it is just provides improvement of gene-editing capabilities that had
existed already for decades, merely allowing them to be implemented more quickly,
cost-effectively and accurately. However, a few recently publishedstudiesshouldgive
the community, and the broader population, pause. In particular, the gene targeting
and stability improvements enabled by the CRISPR-Cas9 system allow for the first
plausibleimplementationofaworkingsynthetic,human-designedgenedrive.
Gene drives are genes that encourage or force their own expression. Simple
examples include targeted nucleases which cut the complementary strand of a
homologous chromosome during replication andforcethecelltoreproduceofcopyof
the only remaining intact gene, or endonucleases that copy themselves into
chromosomes that lack the genes that code for them, but there are many moretypes.
Recall, however, that the CRISPR-Cas system is a nucleic-acid-based system that is
generated by both transcription of CRISPR repeats and spacers and the transcription
of the associated casgenesthataretranslatedtotheCasproteinsthatprocessthem--
indeed, were it not for the protospacer adjacent motif, the CRISPR-Cas machinery
wouldcleaveitselfoutofexistence.
With this is mind, it is not hard to imagine a simple CRISPR-Cas9-based gene
drive: simply insert into the chromosomeaDNAsequencethatwillcodeforachimera
of tracrRNA along with the complementary strand to the gene(s) you would like to
remove/alter; adjacent to this sequence (or not), attach a copy of the gene that codes
for Cas9; et voil. Once this locus is copied, the tracrRNA-crRNA strand will complex
with the Cas9 protein and slice the complementary strand(s). In contrast to previous
gene-deletion methods (in which subsequent reproduction withawild-typeorganism
would give a 50% chance of the offspring inheriting the spliced-out gene), even if the
organism obtains a copy of the deleted gene from a future reproductive partner, the
geneforCas9andguideRNAwouldremain.

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Now, there are many complications with this simple model. As sketched out
above, this gene drive would be vulnerable to drive resistance through random
mutations and repairs that would alter the specificity/affinity of the guide-RNA,
natural sequence polymorphisms, or even the evolution of a method to combat the
nuclease. ButCRISPR-Cas9makesitmoreplausiblethaneverthathumanswillbeable
to make permanent changes to entire populations of organisms. Indeed, a 2016 paper
by Hammond et al. demonstrated a CRISPR-Cas9 gene drive system that rendered
sterile female mosquitos which act as vectors for malaria, with transmission rates to
progeny of 92-99% (depending on the gene locus targeted)[66]. Promising as it may be,
this study alone suggests the need for extreme caution (and perhaps regulation) as
these tools are refined. Released into the wild, on purpose or by accident, dene-drive
mutated organisms have a non-negligible chance of significantly altering the
population at large. While is is straightforward to envision protocols to curb the
unwanted proliferation of organisms the size ofinsectsandlarger,theproliferationof
mutated microscopic organisms will be harder to control. The potential for
unintended adverse effects due to genetic changes with this scope and magnitude is
unprecedented.

Potential
Putting aside severe caution for the moment, it is thrilling to envision the
potential of these systems to perform feats previously thought prohibitively difficult,
or impossible. In the words of van der Oost et al. (2014), in terms of applications of
CRISPR-associated nucleases in general, and Cas9 in particular, the sky seems to be
the limit. For instance, as the CRISPR system has been shown to be found in about
90% of archaea and 40% of bacteria[10]. Though there are few known examples of
archaea that act as pathogens or parasites, examples of bacterial pathogens are
manifold.

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Considering CRISPR-containing bacteria already have the Cas machinery

installed, all that is required for targeted interference in bacterial pathogenesis is a
method for getting a guide-RNA-tracrRNA chimera into the cell -- though both
present a challenge.However,anadvantageofCRISPRsystemsoverpreviousmethods
is the potential impossibility for resistance if the guide-RNA is sufficiently well
designed: one can imagine a guide-RNA that targets a gene that is both
organism-specific and essential; a mutation in the targeted gene that would allow to
resistance to the drug through evasion of of guide-RNA recognition could potentially
cripple the function of an essential protein; any mechanism developed to inhibit the
Cas9 protein responsible for nuclease activity would cripple the organism's own
immunesystem.
In eukaryotes, CRISPRs potentially as a therapeutic agent is currently
hampered in large part by the challenge of getting both the guide-RNA chimera and
the Cas9 enzyme(orthecorrespondingcasgene)intothecell.Accordingly,methodsof
guide-/tracrRNA delivery are a major current focus for cellular and molecular
pharmacology -- truncated guide-RNA results show aglimmerofpromisetothisend:
the shorter the RNA strand to be delivered, the easieritwillbetodesignamethodfor
delivery. However, there are areas in which CRISPR-Cas9 can be put to use more
easily, for instance in cancer gene therapies. Gene editing of T-cells may enable
efficient, targeted antibody-mediated attack of cancer cells, and the ability to shield
antibodies from recognition and subsequent destruction by cancer cells -- immune
cells removed from patients could be quickly and accurately edited andreintroduced.
In fact, a CRISPR-Cas9-based drug that would do exactly that was approved for
clinical trials in the US in June of 2016. In November of 2016, CRISPR-Cas9-edited
immune cells were administered to ahumanpatientforthefirsttimebyateamledby
Dr. Lu YouatSichuanUniversityinChengduaspartofaclinicaltrialforthetreatment
ofaggressivelungcancer.
Though some suggest the excitement about CRISPR is exaggerated, all signs
seem to indicate that pharmaceutical interest in CRISPR-Cas systems as potential

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therapeutics is still only starting to pick up steam -- once the first successful
CRISPR-Cas system passes clinical trials and heads tothemarket,rampantexpansion
in applications for potential CRISPR-Cas-based therapeutics patents can certainly be
expected. It is telling that even in January of 2013, when CRISPR-Cas9 research was
still an emerging field, Bayer AGannounceditwasinvesting300milliondollarsintoa
joint project with a startup, CRISPR Therapeutics, known as Casebia Therapeutics, to
work on breakthroughs in blooddisorders,blindness,andcongenitalheartdisease.As
of early 2017 there are already dozens of CRISPR-focused therapeutics companies
(notable among them: Intellia, Caribou Biosciences, Inc., and ERS genomics), some of
whichhavealreadyhadmultimilliondollarInitialPublicOfferings(IPOs).
In the face of CRISPRs seemingly limitless potential and power, one can only
hope that excitement and wonderment are shown in equal measure to caution and
reverence.

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TopicsinPharmaceuticalSciences

Reviews of the System throughtime:MarraffiniandSondheimer(2010)[17]

Considering CRISPR-containing bacteria already have the Cas machinery

22. Sapranauskas, R., Gasiunas,G.,Fremaux,C.,Barrangou,R.,Horvath,P.,&Siksnys,V.(2011).The

plants using a CRISPR-Cas system. Nature Biotechnology, (August), 14.

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