Cas9-induced large deletions and small indels are controlled in a convergent fashion
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Abstract
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.
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Cell types with deficiencies in DNA damage repair genes are less suited for Cas9 knockouts due to increased risk for genomic instability.
“Cell types with deficiencies in DNA damage repair genes are also less suited for Cas9 knockouts due to their increased risk for genomic instability, which is exacerbated by DSB induction.8”
From Adenine base editor for knockout of proteins: A practical guide from design to analysis with updated MultiEditRbatch (2026) · cites this paper as [8]
The Discussion reports that NHEJ deficiency increases potentially harmful large deletions and complex lesions after Cas9 mutagenesis in mouse embryonic stem cells. But it also reports that Nbn and Polq deficiencies decrease these lesions, so the paper does not support the blanket claim that DNA-repair-deficient cell types are less suited for Cas9 knockouts. (LLM, conf=0.88)
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Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing
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- Authors
- Kosicki, M., Allen, F., Steward, F., Tomberg, K., Pan, Y., & Bradley, A.
- Affiliations
- Lawrence Berkeley National Laboratory (US)
- Wellcome Sanger Institute (GB)
- University of Cambridge (GB)
- Corresponding author
- Bradley, Allan
- Venue
- Nature Communications
- Published
- 2022-06-14
- Volume
- 13
- Issue
- 1
- Pages
- 3422-3422
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC9197861
- OpenAlex
- W4282823317
- Cited by
- 115
- References
- 69
- Citations by year
- 2026: 27 · 2025: 28 · 2024: 26 · 2023: 26 · 2022: 4 · 2021: 2 · 2020: 2
- FWCI
- 7.02
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Apollo (University of Cambridge) (submittedVersion, cc-by)
- Apollo
- Apollo (cc-by)
- Funders
- Wellcome Trust (098051)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- large deletions, small insertions and deletions, CRISPR/Cas9 mutagenesis, non-homologous end joining, niobium nitride, DNA polymerase theta, microhomology-mediated end joining, mouse embryonic stem cells
- MeSH terms
- Animals, DNA Repair, Mice, DNA Breaks, Double-Stranded, INDEL Mutation, DNA End-Joining Repair, CRISPR-Cas Systems
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