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

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2024年4月29日

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Baolei Yuan,
Chongwei Bi,
Yeteng Tian,
Jincheng Wang,
Yiqing Jin,
Khaled Alsayegh,
Muhammad Tehseen,
Gang Yi,
Xuan Zhou,
Yanjiao Shao,
Fernanda Vargas Romero,
Wolfgang Fischle,
Juan Carlos Izpisua Belmonte,
Samir Hamdan,
Yanyi Huang,
Mo Li

摘要

Background

CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.

Results

Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.

Conclusions

Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.