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Cited by 136CRISPR and Genetic EngineeringRNA regulation and diseaseDNA Repair Mechanisms

Microhomologies are prevalent at Cas9-induced larger deletions

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Owens, Dominic D G, Caulder, Adam, Frontera, Vincent, Harman, Joe R, Allan, Alasdair J, Bucakci, Akin, Greder, Lucas, Codner, Gemma F +4 more·2019·Nucleic Acids Research
DOI 10.1093/nar/gkz459PMC PMC6698657
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Abstract

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

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  • Owens et al. found that 23% of clones had large deletions up to 2kb that escaped short-range PCR genotyping after CRISPR-Cas9 deletion in the Runx1 locus of mESCs.

    “Moreover, Owens et al. used CRISPR-Cas9 with two sgRNAs to delete 100-150bp in the Runx1 locus of mESCs and found that 23% of their clones had large deletions (up to 2kb) that escaped genotyping by short-range PCR”

    From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [14]

    In Results, “Cas9D10A nickase generates larger deletions at a similar frequency to Cas9 nuclease” (page unavailable), the paper describes using two sgRNAs to target 100–150 bp at Runx1 in mESCs. It reports deletions extending up to 2 kb that escaped short-range PCR detection. However, 23% (25/108) refers specifically to clones initially scored as homozygous wild type or knock-out and subsequently checked by medium- or long-range PCR, not to all 506 clones analysed. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • MMEJ-derived large deletions are more likely than microhomology-mediated gene conversion in this context.

    “This suggests that MMEJ-derived large deletions (14, 37) are more likely than microhomology-mediated gene conversion in this context, though protein expression has yet to be fully characterised.”

    From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [14]

    The Results section reports that most Cas9-induced larger deletions contain microhomologies, and the Discussion identifies MMEJ as a possible repair pathway. The paper does not compare the likelihood of these deletions with microhomology-mediated gene conversion in the citing paper’s context. Page numbers are not supplied in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Similar damage and frequencies were also observed with the Cas9 D10A nickase.

    “Similar damage and frequencies were also observed with the Cas9 D10A nickase (14).”

    From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [14]

  • Non-negligible frequencies of editing-associated large deletions have been reported after the use of the Cas9 D10A nickase in mESCs and prime editing in early mouse embryos.

    “However, non-negligible frequencies of editingassociated large deletions have been reported after the use of the Cas9 D10A nickase in mESCs (14) and prime editing in early mouse embryos (43).”

    From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [14]

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Authors
Owens, D. D. G., Caulder, A., Frontera, V., Harman, J. R., Allan, A. J., Bucakci, A., Greder, L., Codner, G. F., Hublitz, P., McHugh, P. J., Teboul, L., & de Bruijn, M. F. T. R.
Affiliations
  • University of Oxford (GB)
  • Mary Lyon Centre at MRC Harwell (GB)
  • MRC Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK
Corresponding author
Teboul, Lydia
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2019-05-25
Volume
47
Issue
14
Pages
7402-7417
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkz459
PMC
PMC6698657
OpenAlex
W2945177467
Cited by
136
References
11
Citations by year
2026: 16 · 2025: 12 · 2024: 24 · 2023: 21 · 2022: 25 · 2021: 20 · 2020: 14 · 2019: 4
FWCI
5.21
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institute for Health (U42OD011174)
  • NIHR Oxford BRC and John Fell Fund (131/030, 101/517)
  • U.S. Environmental Protection Agency (CF182, CF170)
  • WIMM Strategic Alliance awards (G0902418, MC_UU_12025)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
CRISPR/Cas9 genome editing, microhomology, microhomology-mediated end joining, Cas9 nickase, single guide RNA, mouse embryo, cultured cells
MeSH terms
Gene Editing, RNA, Guide, CRISPR-Cas Systems, Animals, Cell Line, Chromosome Deletion, Deoxyribonuclease I, Endonucleases, Models, Genetic, Sequence Deletion, RNA, Guide, Kinetoplastida, Chromosomes, Mammalian, Mice, Chromosome Breakpoints, DNA End-Joining Repair, CRISPR-Cas Systems

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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