PT  - JOURNAL ARTICLE
AU  - Yuan, Baolei
AU  - Bi, Chongwei
AU  - Wang, Jinchen
AU  - Jin, Yiqing
AU  - Alsayegh, Khaled
AU  - Tehseen, Muhammad
AU  - Yi, Gang
AU  - Hamdan, Samir
AU  - Huang, Yanyi
AU  - Li, Mo
TI  - Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks
AID  - 10.1101/2022.11.16.516713
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.11.16.516713
4099  - http://biorxiv.org/content/early/2022/11/16/2022.11.16.516713.short
4100  - http://biorxiv.org/content/early/2022/11/16/2022.11.16.516713.full
AB  - 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.Competing Interest StatementA patent application based on methods described in this paper has been filed by King Abdullah University of Science and Technology, in which BY and ML are listed as inventors. The authors declare no other competing interest.