DNA Ends Matter: The Impact of Using CRISPR/Cas9 Variants on DNA Repair Pathway Choice and Editing Profiles at The HBB Locus Tanushree Phadke, Anne Bothmer, Christina Lee, Luis Barrera, Hari Jayaram, Vic Myer, Cecilia Cotta-Ramusino Editas Medicine, 300 Third Street, Cambridge, MA 02142

Introduction Overall modification frequency Deletions across Cas9 variants Modification frequency in the presence of ssODN Donor • Cas9 and its variants can be used to introduce a variety of different DNA cuts including blunt double stranded HBB HBD break (DSB) or dual nicks leaving either a 3’ or 5’ overhang. • The type of cut and donor used can play a role in triggering different repair pathways, thus resulting in various editing outcomes. Gene Correction • Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes at the in the human beta globin gene including indel mutations resulting from Non Characterization of Gene Conversion with D10A Cas9 Characterization of genetic requirements for HDR Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) siRNA using the donor as a template, and, HBB HBD FF Rad51 finally, Gene Conversion (a kind of Rad51

HDR event) using the closely related Vinculin ss-ODN

HBD gene as an endogenous template. siRNA

FF BRCA2 SST-R HR Illustration of the HBB and HBD BRCA2 BRCA2 ? RAD51 loci Gene Conversion β-Actin Gene Correction Gene Conversion

FF- Firefly Luciferase (Negative control)

Insertions across Cas9 variants Model of possible repair outcomes Conclusions Schematic of Cas9 variants DSB 5’ • Different ends generated by

WT Cas9 variants yield different Resection PAM C-NHEJ 3’ repair outcomes, with the 5’ Locus Unaltered overhang yielding more Gene D10A N863A Small Deletions N863A D10A Correction and Gene Small Insertions Alt-NHEJ HDR BRCA2 Conversion. RAD51 PAM PAM • The length of insertions and SD-MMEJ Blunt EJ MMEJ SST-R HR Count deletions seen across Cas9 Insertions Deletions Correction Conversion variants is substantially different C-NHEJ: Canonical Non-Homologous End Joining suggesting the role of multiple PAM PAM Alt-NHEJ: Alternative Non-Homologous End Joining repair pathways. SD-MMEJ : Synthesis Dependent Microhomology Mediated End Joining MMEJ : Microhomology Mediated End Joining • Gene Conversion appears to be Insertion lengths (nts) Blunt-EJ: Blunt End Joining SST-R: Single Strand Template Repair RAD51 and BRCA2 dependent, HR: Homologous Recombination but Gene Correction does not.