576. DNA Ends Matter: The Impact of Using CRISPR/Cas9 Variants on DNA Repair Pathway Choices and Editing Profiles at the HBB Locus

Sickle Cell Anemia is an inherited recessive disorder caused by a single point mutation in the human beta globin (HBB) gene resulting in an abnormal type of hemoglobin. Here we report targeting the HBB locus using CRISPR/Cas9 technology for correction of Sickle Cell Disease, which affects nearly 1 million people worldwide. Cas9 and its variants can be used to introduce a variety of breaks including blunt double stranded break (DSB), single nicks, 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 profiles. Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes including indel mutations resulting from Non Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) using the donor as a template, and, finally, Gene Conversion (a kind of HDR event) using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene that corrects the sickle cell point mutation. We observed that the Cas9 nickase/gRNA pair leaving a 5’ overhang displayed a significantly higher frequency of gene conversion and gene correction than other Cas9-induced DNA end structures. We also provide evidence that overexpression or down-regulation of critical factors in the repair pathways influence the repair pathway balance.In summary, we demonstrate that the frequency of various repair outcomes under different conditions offers insight into the mechanisms of repair of Cas9-induced DNA cleavage. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.