Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks

CRISPR-Cas9, an efficient genome editing tool, has been widely used in research and holds great promise in the clinic. However, large unintended rearrangements of the genome occur frequently after CRISPR-Cas9 editing and their potential risk cannot be ignored. In this study, we detected large deletions (LDs) induced by CRISPR-Cas9 in human embryonic stem cells (hESCs) and found the microhomology end joining (MMEJ) DNA repair pathway plays a predominant role in LD. We genetically targeted PARP1, RPA, POLQ and LIG3, which play critical roles in MMEJ, during CRISPR-Cas9 editing. By analyzing LD events in two independent gene loci, CD9 and PIGA, using flow cytometry and long-read individual molecule sequencing (IDMseq), we showed that knocking down PARP1 and LIG3 does not alter the frequency of Cas9-induced LD, while knocking down or inhibiting POLQ dramatically reduces LD. Knocking down RPA increases LD frequency, and overexpression of RPAs consistently reduces LD frequency. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically increase the efficiency of homology-directed repair (HDR). In conclusion, RPA and POLQ play opposite roles in Cas9-induced LD, modulation of POLQ and RPA can reduce LD and improve HDR, thus holding promise for safe and precise genome editing.