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bioRxiv · 10.1101/2022.11.16.516713

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

Yuan, B.·Bi, C.·Wang, J.·Jin, Y.·Alsayegh, K.·Tehseen, M.·Yi, G.·Hamdan, S.·Huang, Y.·Li, M.
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Abstract

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.

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Yuan, B., Bi, C., Wang, J., Jin, Y., Alsayegh, K., Tehseen, M., Yi, G., Hamdan, S., Huang, Y., Li, M.. 2022-11-16. Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks. https://doi.org/10.1101/2022.11.16.516713

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