Effect of Different CRISPR/Cas9 Variants on Repair Pathway Choice

Transcription

1 Effect of Different CRISPR/Cas9 Variants on Repair Pathway Choice Cecilia Cotta-Ramusino 1

2 Cas9 Stimulates the Endogenous Repair Pathways WT Cas9 DSB C-NHEJ Resection 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

3 Cas9 is a Flexible Tool WT Cas9 N863A Nickases D10A Nickases Blunt 3 3 Overhang Overhang Could we engage different pathways by using these different variants? Could we selectively stimulate HDR? 3

4 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

5 Modification (%) DSBs Generated by D10A are Predominantly Repaired by HDR 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% HDR Insertions Deletions Long insertions Repetitions of the overhang Microhomology mediated Alt-NHEJ 0% WT N863A D10A WT N863A D10A SD-MMEJ 3 Bothmer et al., Nat Comm 20175

6 Modification (%) DSBs Generated by D10A are Predominantly Repaired by HDR 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% HDR Insertions Deletions 0% WT N863A D10A WT N863A D10A 3 6

7 Modification (%) DSBs Generated by D10A are Predominantly Repaired by HDR 100% 90% 80% 70% Gene Correction Insertions Deletions 60% 50% 40% 30% HBB HBD ssodn 20% 10% 0% WT N863A D10A WT N863A D10A Gene Correction 3 Bothmer et al., Nat Comm 20177

8 Do and Gene Correction have the same Genetic Requirement? Gene Correction HBB HBD ssodn Do they both dependent on the HR pathway? 8

9 Gene Correction and Gene Correction have Different Genetic Requirements Neg. Cont. K.D. Neg. Cont. K.D. Neg. Cont. K.D. SS ODN donor Endogenous HBD Plasmid donor 9

10 Gene Correction and Gene Correction have Different Genetic Requirements Neg. Cont. K.D. Neg. Cont. K.D. Neg. Cont. K.D. SS ODN donor Endogenous HBD Plasmid donor HR is required for repair from double stranded donors (endogenous homology tracks or plasmids) but not single stranded donors 10

11 3 Overhang is Required to Promote Gene Conversion and Gene Correction Overhang processing generation Alt-NHEJ ss-odn 3? SST-R Gene Correction HR BRCA2 11

12 Fold change in Gene HDR Correction (%) Rad52 has a Role in Promoting SST-R Fold change in Gene Correction HDR (%) K.O. of Rad52 Overexpression of Rad *** control (ko) Neg. Cont. K.O. Rad52 Rad52 (ko) 0.0 Neg. Cont. control O.E. Rad52 Rad52 Anne Bothmer Poster

13 Conclusions from the Dual Nick Analysis Different ends activate different DNA repair pathways WT Cas9 C-NHEJ N863A Nickases Alt-NHEJ D10A Nickases HDR Different donors stimulate different pathways Gene Correction mediated by ssodn is not HR dependent but partially depends on Rad52 Bothmer et al., Nat Comm

14 Characterization of the DNA Repair Pathway in Response to Single Nick D10A N863A 14

15 D10A Nick Results in More Frequent HDR Than N863A % of Modifications Gene Correction Insertions Deletions **** D10A N863A 15

16 D10A but Not N863A Nick Repair Depends on HR Pathway % of Modifications Gene Correction Insertions Deletions c sirna N.C. N.C. D10A N863A 16

17 D10A but Not N863A Nick Repair Depends on HR Pathway % of Modifications Gene Correction Insertions Deletions sirna N.C. N.C. D10A N863A 17

18 Working Model D10A N863A Nick Protected Removal of the Cas9/gRNA in S phase Nick exposed Nick DSB More easily repaired Single Nick Repair Simple Ligation 18

19 Working Model D10A N863A Nick Protected Removal of the Cas9/gRNA in S phase Nick exposed Nick DSB More easily repaired Single Nick Repair Simple Ligation 19

20 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

21 Thank you! 21