Abstract
CRISPR-Cas9, an efficient genome editing tool, has been widelyused in research and holds great promise in the clinic. However, thelarge unintended rearrangements of the genome after CRISPR-Cas9editing occur frequently and their potential risk cannot be ignored. In this study, we detected large deletions (LDs) induced by Cas9 inhuman embryonic stem cells (hESCs) and found micro-homologyend joining (MMEJ) repair pathway plays a predominant role inLD. We genetically targeted PARP1, RPA, PolQ and Lig3, whichplays critical roles in MMEJ, during CRISPR-Cas9 editing. Wefound that knocking down PARP1 and Lig3 does not alter LDfrequency, while knocking down or inhibiting PolQ dramaticallyreduces Cas9-induced LD frequency. Knocking down RPA in-creases LD frequency, and consistently, overexpression of RPAsreduces the frequency of LD. In conclusion, RPAs and PolQ playopposite roles in Cas9-induced LD and may be promising targets forreducing large rearrangement frequency during genome editing.