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CRISPR RiboNucleoProtein

Transfection
Dr. Sandra Lagauzère
- Lipocalyx (Germany)
RNA-guided engineered nucleases for genome editing

Non-Homologous End Joining Homology Directed Repair


Potential Desired modifications

 Mutations  Gene knock-out


 Insertions /  Gene tagging
deletions  Point mutations
 Gene knock-out  Knock-in
 Promotor study
Other applications possible: e.g. CRISPRi/a with dCas9
For efficient genome editing,
successful delivery of gRNA and Cas9 into cells is essential
…but challenging!

long phosphate
large protein 160kDa backbone
(high negative charge)

1. Protection of CRISPR components


2. Crossing the cell membrane
3. Release into the cytosol
4. Penetrating the nucleus
Animal experiments: direct injection in 1-cell embryos  gene-edited animals

In vitro cell cultures: transfection of plasmids using lipofection or electroporation


Indirect methods to instruct the cells Straightforward
 Stable and inexpensive
 Easy-to-use
 Can include selectable markers
 Standard transfection or viral
transduction
 Stable and inexpensive
 Easy-to-use
 Can include selectable markers
 Standard transfection or viral
transduction

 Lag of expression (>12h) and


long persistence (weeks)
 Difficult to regulate
 High “off-target” effects
 pDNA transfection stressful
(primary cells) or inefficient
(immune cells)
 Stable and inexpensive  Cheap if use of IVT RNAs
 Easy-to-use  Standard transfection
 Can include selectable markers  Fast and transient expression
 Standard transfection or viral  Less off-target effects
transduction

 Lag of expression (>12h) and


long persistence (weeks)
 Difficult to regulate
 High “off-target” effects
 pDNA transfection stressful
(primary cells) or inefficient
(immune cells)
 Stable and inexpensive  Cheap if use of IVT RNAs
 Easy-to-use  Standard transfection
 Can include selectable markers  Fast and transient expression
 Standard transfection or viral  Less off-target effects
transduction

 Lag of expression (>12h) and  Expensive with purchase of


long persistence (weeks) synthetic RNAs
 Difficult to regulate  mRNA not stable
 High “off-target” effects  Co-transfection of gRNA and
 pDNA transfection stressful mRNA can create a lag at peak
(primary cells) or inefficient expression
(immune cells)
 Most transient expression and
 Stable and inexpensive  Cheap if use of IVT RNAs least delay for therapeutics
 Easy-to-use  Standard transfection  Easy to adjust Cas9 and gRNA
 Can include selectable markers  Fast and transient expression amounts
 Standard transfection or viral  Less off-target effects  Rapid protein clearance
transduction  Very limited off-target effects

 Lag of expression (>12h) and  Expensive with purchase of


long persistence (weeks) synthetic RNAs
 Difficult to regulate  mRNA not stable
 High “off-target” effects  Co-transfection of gRNA and
 pDNA transfection stressful mRNA can create a lag at peak
(primary cells) or inefficient expression
(immune cells)
 Most transient expression and
 Stable and inexpensive  Cheap if use of IVT RNAs least delay for therapeutics
 Easy-to-use  Standard transfection  Easy to adjust Cas9 and gRNA
 Can include selectable markers  Fast and transient expression amounts
 Standard transfection or viral  Less off-target effects  Rapid protein clearance
transduction  Very limited off-target-effects

 Lag of expression (>12h) and  Expensive with purchase of  Expensive


long persistence (weeks) synthetic RNAs  Challenging delivery: most
 Difficult to regulate  mRNA not stable difficult format
 High “off-target” effects  Co-transfection of gRNA and  can trigger immune response if
 pDNA transfection stressful mRNA can create a lag at peak dosage not carefully monitored
(primary cells) or inefficient expression
(immune cells)
Animal experiments: direct injection in 1-cell embryos  gene-edited animals

In vitro cell cultures: transfection of plasmids using lipofection or electroporation


Glass et al. "Engineering the delivery system for CRISPR-based genome editing." Trends in biotechnology (2018).
Modification of the target cell Modification of the deliverable

- Max capacity 5kb - One-cell - Not suitable in vivo Encapsulation or complexation with
- Concern for use in application - Also in cell cultures peptides, liposomes, cationic lipids,
clinical settings of muscle or nerve gold nanoparticles, carbon nanotubes…
cells reacting to
electrical pulses
and polymers?
Fusion peptide
The VIROMER® Technology

Polymer
Model: HeLa cells, Viromer® GREEN, labelled siRNA

1h early endocytosis
uptake

3h late endocytosis
Accumulation
near the
nucleus

4.5h cargo release


Discharge from
endosomes,
starting diffusion
Cells Target gene Info Reference

hMDMC BATF CRISPRi with dCas9 Gupta et al. "IL-6 augments IL-4-induced polarization of primary
macrophages human macrophages through synergy of STAT3, STAT6 and BATF
transcription factors."
OncoImmunology 7.10 (2018): e1494110.
HGT-1 TAS2R43 KO-stable cells Liszt et al. "Caffeine induces gastric acid secretion via bitter taste
human gastric cancer signaling in gastric parietal cells."
cells PNAS 114.30 (2017): E6260-E6269.
MDFs RIPK1 KO-stable cells Liccardi G. et al. "RIPK1 and Caspase-8 ensure chromosome
Mouse dermal stability independently of their role in cell death and
fibroblasts inflammation."
Molecular cell (2018).
HAP1 UNG KO-stable cells Sarno A. et al. "Uracil–DNA glycosylase UNG1 isoform variant
Human fibroblast-like supports class switch recombination and repairs nuclear genomic
cells derived from CML uracil." Nucleic Acids Research (2019).
KBM7 cells
Co-transfection of mRNA-Cas13a and gRNA

Modified mRNA encoding Cas13a ribonuclease


- reduces and prevents infection of Influenza virus and human respiratory syncytial virus in MDCK and A549 cells
- prevents spread to non-infected cells

Electroporation

Viromer® RED

LipofectamineTM 3000

Bawage et al. "Synthetic mRNA expressed Cas13a mitigates RNA virus infections." bioRxiv (2018): 370460.
Design of a new reagent for CRISPR / RNP delivery -
Viromer Chemical Space

alkyl substitutions carboxy-alkyl substitutions


# of carbons 3-12 # of carbons 3-16
cyclic / linear Y/N aromaticity Y/N
aromatic Y/N % substitution 0-100
% substitution 0- 100

6.000 structures synthesized and


tested for RNP transfection

type of polyamine
branching
size

Screening based on RFP expression KO in stable HEK cells


Selection leads with >60% reduction RFP and no toxicity
(in comp. <20% with Lipofectamine CRISPRMAX)
Screening Delivery >80% of RNP delivery
>90% survival

Selection of 1 lead

Validation and optimization


were performed in model cell
lines A549, HEK293,HeLa, C2C12
> KO of HPRT1

cut

50-90% of targeted editing events


(NHEJ mutations estimated by T7 assay and/or sequencing) Editing
Final reagent is easily scalable and adjustable

Example of transfection protocol optimization in A549

2x RNP start conc. 3x transfer volumes


1.25 or 2.5µM of transfection mix
Optimal frame
Optimal frame in wells
Starts being
Starts being toxic
toxic

resulting in 6.25 to 37.5nM final RNP

Lower
Lower efficiency
efficiency
First external data are coming…
Safe delivery and up to 50% of targeted editing in C2C12 myoblasts
50-60% of editing events in guinea pig primary fibroblasts

Data courtesy of Dr. Fritz Benseler, MPI Goettingen, Germany


Excess of gRNA leads to >80% of editing in HUVECs

Slightly toxic
RNP delivery in mouse pancreatic ductal adenocarcinoma cells (PDAC)

Viromer

Test of 3 different crRNA annealed with a labelled tracrRNA (ATTO647, from IDT)

For all 3 gRNAs, both reagents deliver RNP complex in almost 100% of cells
MFI substantially higher with Viromer
New FACS sorting with crRNA#1 – test of 2 different RNP concentrations

FOLLOWING STEPS…

- Surveyor assay with crRNA#1 and 50nM RNP  around 30% of positive candidates
- Sanger sequencing  confirms targeted homozygote editing for 50% of cells.

“About the cell line I remember that these PDAC cells are not very easy
to transfect with DNA plasmids: I did a transfection with plasmid DNA
once using a plasmid coding for all the CRISPR system and orange
protein as reporter but I got very low efficiency.”

Data courtesy of Francesco De Sanctis, University of Verona, Dept Medicine, Sect. Immunology (Italy).
+ HDR template?
Homology Directed Repair
and
HDR template
ssDNA

Small insertions with ssODNs (50-200bp)


Now also possible to have long ssDNA (up 2000bp)

Delivery

- co-transfection
- complexation
and
HDR template
ssDNA

Small insertions with ssODNs (50-200bp)


Now also possible to have long ssDNA (up 2000bp)

Delivery

- co-transfection
With a polymer-based
- complexation nanoparticle system
GeneEdit, Berkeley

Lee et al. "Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair.
" Nature biomedical engineering 1.11 (2017): 889.
CRISPR-Gold in vitro Mutants with
Restoration of dystrophin no Dystrophin
expression in primary
myoblasts from mutants

CRISPR-Gold in vivo
Successful editing after
muscle injection
Delivery

- co-transfection
- complexation
+ HDR template
ssDNA

Delivery

- co-transfection ?
- complexation
Use of most robust in-house assay: A549 cells, target HPRT1 gene (InDel 70%)

ssODN: non-PAM with 40nt homology arm length, size: 84nt, restriction site HindIII

Strategy 1 Strategy 2

Co-transfection of RNP + donor 2 sequential transfections (donor > RNP)


Standard transfection Standard conditions
+ test of x2 and x3 Viromer® (NP612)

+/- enhancer +/- enhancer

Ratio: 12.5nM RNP / 4nM donor


Sequential
Co-transfection
transfection

uncut

cut

96% 65% 78% 81% 84% 81% 58% 70% 67% 82% 71% 81% 84% 87% 72% 99% InDel
Sequential
Co-transfection
transfection

uncut

cut

96% 65% 78% 81% 84% 81% 58% 70% 67% 82% 71% 81% 84% 87% 72% 99 % InDel

non
HDR
cut

10% 12% 12% 7% 7% 6% 19% 24% 11% 12% 9% 6% 6% 10% 12% 13% HDR
Sequential
Co-transfection
transfection

uncut

cut

96% 65% 78% 81% 84% 81% 58% 70% 67% 82% 71% 81% 84% 87% 72% 99 % InDel

non
HDR
cut

10% 12% 12% 7% 7% 6% 19% 24% 11% 12% 9% 6% 6% 10% 12% 13% HDR
Conclusion

- Different formats: plasmid > mRNA > RNP


 choice will depend on cells, lab capacities and expertise, costs
 RNP: most transient Cas9 activity, most straightforward workflow

- Many delivery methods: injection, virus, electroporation, chemical transfection


 but not for in vivo

- Chemical transfection advantages: easy-to-use, adjustable, scalable

- Polymer nanoparticles Viromer® >> lipofection:


 less toxic, no interaction with cell physiology, lower effects downstream
Thanks for your attention!