Slide 1: Effect of Different CRISPR/Cas9 Variants on Repair Pathway Choice
Effect of Different CRISPR/Cas9 Variants on Repair Pathway Choice Cecilia Cotta-Ramusino www.editasmedicine.com 1
Slide 2: Cas9 Stimulates the Endogenous Repair Pathways
Cas9 Stimulates the Endogenous Repair Pathways 5’ WT Cas9 DSB 5’ Resection C-NHEJ 3’ Locus Unaltered Small Deletions Small Insertions Alt-NHEJ HDR 3’ Blunt EJ MMEJ SD-MMEJ Deletions Deletions Insertions SSA HR Large Deletions Correction 2
Slide 3: Cas9 is a Flexible Tool
Cas9 is a Flexible Tool WT Cas9 N863A Nickases D10A Nickases 5’ 5’ 5’ 3’ 5’ Blunt 3’ Overhang 5’ Overhang • Could we engage different pathways by using these different variants? • Could we selectively stimulate HDR? 3
Slide 4: Evaluating DNA Repair Pathways Engaged in Response to Lesions Induced by Cas9 Variants
Evaluating DNA Repair Pathways Engaged in Response to Lesions Induced by Cas9 Variants Sickle Cell Disease: Editing of the HBB locus HBB HBD ~7kb 4
Slide 5: DSBs Generated by D10A are Predominantly Repaired by HDR
DSBs Generated by D10A are Predominantly Repaired by HDR HDR 100% Insertions 90% Deletions Modification (%) 80% 70% 60% 50% • Long insertions • Repetitions of the overhang 40% • Microhomology mediated 30% 20% 10% Alt-NHEJ 0% SD-MMEJ WT WT N863A N863A D10A D10A 3’ 5’ Bothmer et al., Nat Comm 20175
Slide 6: DSBs Generated by D10A are Predominantly Repaired by HDR
DSBs Generated by D10A are Predominantly Repaired by HDR HDR 100% Insertions 90% Deletions Modification (%) 80% 70% 60% 50% 40% 30% 20% 10% 0% WT WT N863A N863A D10A D10A 3’ 5’ 6
Slide 7: DSBs Generated by D10A are Predominantly Repaired by HDR
DSBs Generated by D10A are Predominantly Repaired by HDR Gene Conversion Gene Correction 100% Insertions 90% Deletions Modification (%) 80% 70% 60% HBB HBD 50% 40% 30% ssODN 20% Gene Conversion 10% 0% WT WT N863A N863A D10A D10A Gene Correction 3’ 5’ Bothmer et al., Nat Comm 20177
Slide 8: Do Gene Conversion and Gene Correction have the same Genetic Requirement?
Do Gene Conversion and Gene Correction have the same Genetic Requirement? Gene Correction Gene Conversion HBB HBD ssODN Do they both dependent on the HR pathway? 8
Slide 9: Gene Conversion and Gene Correction have Different Genetic Requirements
Gene Conversion and Gene Correction have Different Genetic Requirements Gene Conversion Gene Conversion Gene Correction Neg. K.D. Neg. K.D. Neg. K.D. Cont. Rad51 Cont. Rad51 Cont. Rad51 SS ODN donor Endogenous HBD Plasmid donor 9
Slide 10: Gene Conversion and Gene Correction have Different Genetic Requirements
Gene Conversion and Gene Correction have Different Genetic Requirements Gene Conversion Gene Conversion Gene Correction Neg. K.D. Neg. K.D. Neg. K.D. Cont. Rad51 Cont. Rad51 Cont. Rad51 SS ODN donor Endogenous HBD Plasmid donor HR is required for repair from double stranded donors (endogenous homolo
Slide 11: 3' Overhang is Required to Promote Gene Conversion and Gene Correction
3’ Overhang is Required to Promote Gene Conversion and Gene Correction 5’ Overhang 5’ 5’ 5’ processing 3’ 3’ 3’ generation 3’ Alt-NHEJ ss-ODN SST-R HR BRCA2 ? Rad51 Gene Correction Gene Conversion 11
Slide 12: Rad52 has a Role in Promoting SST-R
Rad52 has a Role in Promoting SST-R K.O. of Rad52 Overexpression of Rad52 2.0 2.0 HDRCorrection Correction Fold change in Fold change in 1.5 1.5 *** (%) HDR (%) 1.0 1.0 Gene Gene 0.5 0.5 0.0 0.0 Neg. K.O. ) o) ko Neg. O.E. (k ol 52 l( r 52 ad Cont. Rad52 nt ro Cont. Rad52 ad co R nt co R Anne Bothme
Slide 13: Conclusions from the Dual Nick Analysis
Conclusions from the Dual Nick Analysis • Different ends activate different DNA repair pathways 5’ WT Cas9 C-NHEJ 5’ 5’ N863A Nickases D10A Nickases Alt-NHEJ HDR • Different donors stimulate different pathways Gene Correction mediated by ssODN is not HR dependent but partially depends on Rad52 Bothm
Slide 14: Characterization of the DNA Repair Pathway in Response to Single Nick
Characterization of the DNA Repair Pathway in Response to Single Nick D10A N863A 14
Slide 15: D10A Nick Results in More Frequent HDR Than N863A
D10A Nick Results in More Frequent HDR Than N863A Gene Conversion Gene Correction Insertions Deletions % of Modifications **** D10A N863A 15
Slide 16: D10A but Not N863A Nick Repair Depends on HR Pathway
D10A but Not N863A Nick Repair Depends on HR Pathway Gene Conversion Gene Correction Insertions % of Modifications Deletions c siRNA N.C. Rad51 N.C. Rad51 D10A N863A 16
Slide 17: D10A but Not N863A Nick Repair Depends on HR Pathway
D10A but Not N863A Nick Repair Depends on HR Pathway Gene Conversion Gene Correction Insertions % of Modifications Deletions siRNA N.C. Rad51 N.C. Rad51 D10A N863A 17
Slide 18: Working Model
Working Model D10A N863A Nick “Protected” Nick exposed Removal of the Cas9/gRNA in S phase More easily repaired • Single Nick Repair Nick DSB • Simple Ligation 18
Slide 19: Working Model
Working Model D10A N863A Nick “Protected” Nick exposed Removal of the Cas9/gRNA in S phase More easily repaired • Single Nick Repair Nick DSB • Simple Ligation 19
Slide 20: Overall Conclusions
Overall Conclusions • Different lesions activate different repair pathways • Different donors activate different repair pathways • Understanding the differential pathway engagement allows for a deterministic approach in designing research and therapeutic genome engineering strategies 20
Slide 21: Thank you!
Thank you! www.editasmedicine.com 21