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Transfection
Dr. Sandra Lagauzère
- Lipocalyx (Germany)
RNA-guided engineered nucleases for genome editing
long phosphate
large protein 160kDa backbone
(high negative charge)
- 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
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
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
type of polyamine
branching
size
Selection of 1 lead
cut
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
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
Delivery
- co-transfection
- complexation
and
HDR template
ssDNA
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
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