Cas9-induced large deletions and small indels are controlled in a convergent fashion

• Published in: Nature communications Vol. 13; no. 1; pp. 3422 - 11
• Main Authors: Kosicki, Michael, Allen, Felicity, Steward, Frances, Tomberg, Kärt, Pan, Yangyang, Bradley, Allan
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14.06.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/337/1427/2122; 631/337/4041/3196; Animals; BASIC BIOLOGICAL SCIENCES; Clonal deletion; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA End-Joining Repair - genetics; DNA repair; DNA Repair - genetics; Embryo cells; Exonuclease; Gene deletion; Genomics; Homology; Humanities and Social Sciences; INDEL Mutation; Mice; multidisciplinary; Mutagenesis; Non-homologous end joining; Repair; Science; Science (multidisciplinary); Stem cell transplantation; Stem cells
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-30480-8

Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq . Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation. CRISPR/Cas9 system has revolutionized science and therapy, but DNA damage it causes often goes beyond the desired ’precision editing’. Here, the authors identify general and target specific DNA repair pathways responsible for unwanted mutagenesis.