Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements
Open accessAbstract
CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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Inaccurate (off-target) genome editing occurs when CRISPR-induced DNA cleavage and repair happens at genomic locations not intended for modification, typically sites close in sequence to the intended editing site.
“Inaccurate (off-target) genome editing occurs when CRISPR-induced DNA cleavage and repair happens at genomic locations not intended for modification, typically sites that are close in sequence to the intended editing site (reviewed in ref. 93).”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [93]
The Abstract distinguishes targeted sites from distal off-target sequences, and its discussion of prior assessments mentions potential off-target sites. These passages support the distinction between intended and off-target editing, but do not say that off-target sites are typically close in sequence to the intended site (no page number is available in our copy). (LLM, conf=0.78)
Experimental — accuracy not yet measuredSubstantial deletions or rearrangements of chromosomes have also been observed in CRISPR editing due to repairs facilitated by mechanisms such as homology-directed repair or single-strand annealing within cells.
“Nonetheless, substantial deletions or rearrangements of chromosomes have also been observed in CRISPR editing due to repairs facilitated by mechanisms such as homology-directed repair or single-strand annealing within cells.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [56]
The Abstract reports large deletions and complex genomic rearrangements after CRISPR–Cas9 editing in the cell types studied. A further passage, indexed under Abstract, suggests inter-homologue strand invasion for two recombinant alleles, but the paper does not establish homology-directed repair or single-strand annealing as the mechanisms causing the substantial deletions or rearrangements claimed here. (LLM, conf=0.86)
Experimental — accuracy not yet measuredKosicki et al. observed large deletions up to 6kb and other complex genomic lesions at frequencies of 5-20% of clones after targeting PigA and Cd9 loci in mESC lines and primary mouse cells, and PIGA in human RPE cells.
“Consistent with this, Kosicki and colleagues observed large deletions (up to 6kb) and other complex genomic lesions at frequencies of 5-20% of their clones after targeting the PigA and Cd9 loci in two mouse embryonic stem cell (mESC) lines and primary mouse cells from the bone marrow, as well as the PIGA gene in immortalised human female retinal pigment epithelial cells (12).”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [12]
The paper reports deletions up to 6 kb in PigA-deficient mouse ES-cell clones and documents complex lesions at PigA, Cd9 and human RPE1 PIGA (passages labeled Abstract; no page supplied). But 5–20% refers to PigA loss after particular intronic guides, not the frequency of these lesions across all clones and cell types. In primary mouse bone-marrow cells, the target was GFP, and large deletions were found in 35 of 96 selected clones—not 5–20%. (LLM, conf=0.91)
Experimental — accuracy not yet measuredRepair of DSBs introduced by Cas9 has been shown to be error prone and may result in complex rearrangements, including chromothripsis, at the site of repair.
“The repair of DSBs introduced by Cas9 has been shown to be error prone and may result in complex rearrangements, including chromothripsis, at the site of repair25–27.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [26]
The Abstract reports on-target mutagenesis and complex genomic rearrangements following Cas9-induced breaks, supporting the claim that repair can be error prone. Our copy does not identify chromothripsis as one of those outcomes, so that specific example is not supported by this source. (LLM, conf=0.87)
Experimental — accuracy not yet measuredVariability of NHEJ-mediated repair outcomes plagues non-coding CRISPR screens with unprogrammed editing outcomes.
“Additional challenges include that the variability of NHEJ-mediated repair outcomes plagues these screens with unprogrammed editing outcomes204,206”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [206]
The paper reports diverse, unprogrammed outcomes after Cas9 cutting, including large deletions, inversions and lesions away from the cut site; it also finds that intronic guides can cause unexpected gene loss (paper text labeled Abstract; page unavailable). It does not establish that NHEJ specifically causes these outcomes or directly assess their impact on non-coding CRISPR screens. (LLM, conf=0.84)
Experimental — accuracy not yet measured
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Self-citationTools for efficient genome editing; ZFN, TALEN, and CRISPR
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Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins
Lee, J., Mou, H., Ibraheim, R., Liang, S.-Q., Liu, P., Xue, W., & Sontheimer, E. J. (2019) · RNA · doi:10.1261/rna.071704.119 · Cited by 100 · see citing works
Paralog dependency indirectly affects the robustness of human cells
Dandage, R., & Landry, C. R. (2019) · Molecular Systems Biology · doi:10.15252/msb.20198871 · Cited by 65 · see citing works
Donohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.07.035 · Cited by 69 · see citing works
Single and multiple gene knockouts by CRISPR–Cas9 in maize
Doll, N. M., Gilles, L. M., Gérentes, M.-F., Richard, C., Just, J., Fierlej, Y., Borrelli, V. M. G., Gendrot, G., Ingram, G. C., Rogowsky, P. M., et al. (2019) · Plant Cell Reports · doi:10.1007/s00299-019-02378-1 · Cited by 71 · see citing works
Renken, S., Nakajima, T., Magalhaes, I., Mattsson, J., Lundqvist, A., Arnér, E. S. J., Kiessling, R., & Wickström, S. L. (2022) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2021-004458 · Cited by 67 · see citing works
One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids
Geurts, M. H., Gandhi, S., Boretto, M. G., Akkerman, N., Derks, L. L. M., van Son, G., Celotti, M., Harshuk-Shabso, S., Peci, F., Begthel, H., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-40701-3 · Cited by 66 · see citing works
Genome-wide detection of CRISPR editing in vivo using GUIDE-tag
Liang, S.-Q., Liu, P., Smith, J. L., Mintzer, E., Maitland, S., Dong, X., Yang, Q., Lee, J., Haynes, C. M., Zhu, L. J., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28135-9 · Cited by 54 · see citing works
Kondo, K., & Taguchi, C. (2022) · Food Safety · doi:10.14252/foodsafetyfscj.d-21-00016 · Cited by 49 · see citing works
Unexpected CRISPR on-target effects
Lee, H., & Kim, J.-S. (2018) · Nature Biotechnology · doi:10.1038/nbt.4207 · Cited by 46 · see citing works
Emerging applications of gene editing technologies for the development of climate-resilient crops
Chavhan, R. L., Jaybhaye, S. G., Hinge, V. R., Deshmukh, A. S., Shaikh, U. S., Jadhav, P. K., Kadam, U. S., & Hong, J. C. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1524767 · Cited by 43 · see citing works
“…coRanntrolled editing system ruling out adverse editing outcomes, especially large deletions, chromosomal rearrangements, and even chromosome loss (Kosicki et al., 2018; Cullot et al., 2019; Leibowitz et al., 2021).,Enhancement of HDR methods pertains touse of asymmetric ssODN templates…”
Xiong, X., Li, Z., Liang, J., Liu, K., Li, C., & Li, J.-F. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac166 · Cited by 41 · see citing works
Directed evolution of hyperactive integrases for site specific insertion of transgenes
Hew, B. E., Gupta, S., Sato, R., Waller, D. F., Stoytchev, I., Short, J. E., Sharek, L., Tran, C. T., Badran, A. H., & Owens, J. B. (2024) · Nucleic Acids Research · doi:10.1093/nar/gkae534 · Cited by 35 · see citing works
Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases
Carlson-Stevermer, J., Das, A., Abdeen, A. A., Fiflis, D., Grindel, B. I., Saxena, S., Akcan, T., Alam, T., Kletzien, H., Kohlenberg, L., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-20065-8 · Cited by 32 · see citing works
Li, J., Wu, P., Cao, Z., Huang, G., Lu, Z., Yan, J., Zhang, H., Zhou, Y., Liu, R., Chen, H., et al. (2024) · Cell Reports · doi:10.1016/j.celrep.2024.113765 · Cited by 26 · see citing works
Development of multiplexed orthogonal base editor (MOBE) systems
Cowan, Q. T., Gu, S., Gu, W., Ranzau, B. L., Simonson, T. S., & Komor, A. C. (2025) · Nature Biotechnology · doi:10.1038/s41587-024-02240-0 · Cited by 26 · see citing works
Rapid interrogation of cancer cell of origin through CRISPR editing
Feng, W., Cao, Z., Lim, P. X., Zhao, H., Luo, H., Mao, N., Lee, Y. S., Rivera, A. A., Choi, D., Wu, C., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2110344118 · Cited by 25 · see citing works
Garrigues, S., Peng, M., Kun, R. S., & de Vries, R. P. (2023) · mBio · doi:10.1128/mbio.00668-23 · Cited by 22 · see citing works
Carvalho, C., Lemos, L., Antas, P., & Seabra, M. C. (2023) · Frontiers in Ophthalmology · doi:10.3389/fopht.2023.1270561 · Cited by 21 · see citing works
Development and application of prime editing in plants
Tingting, L., Jinpeng, Z., Xi, Y., Kejian, W., Yuchun, R., & Chun, W. (2023) · Rice Science · doi:10.1016/j.rsci.2023.07.005 · Cited by 21 · see citing works
Genome-wide CRISPR off-target DNA break detection by the BLISS method
Ballarino, R., Bouwman, B. A. M., & Crosetto, N. (2021) · Methods in molecular biology · doi:10.1007/978-1-0716-0687-2_15 · Cited by 8 · see citing works
- Authors
- Kosicki, M., Tomberg, K., & Bradley, A.
- Affiliations
- Wellcome Sanger Institute (GB)
- Corresponding author
- Bradley, Allan
- Venue
- Nature Biotechnology
- Published
- 2018-07-16
- Volume
- 36
- Issue
- 8
- Pages
- 765-771
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6390938
- OpenAlex
- W2883697525
- Cited by
- 1,883
- References
- 38
- Citations by year
- 2026: 227 · 2025: 177 · 2024: 199 · 2023: 215 · 2022: 209 · 2021: 244 · 2020: 264 · 2019: 254 · 2018: 94
- FWCI
- 76.10
- Type
- journal article
- Language
- en
- Update notices
- erratum (2018-10-01)
- Open access
- green
- europepmc.org
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Wellcome Trust (079643, 098051)
- 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, DNA double-strand break repair, large deletions, genome rearrangement, crossover events, single guide RNA, mouse embryonic stem cells, hematopoietic progenitor cells, gene editing specificity, off-target effects
- MeSH terms
- Animals, Genotype, Humans, Polymerase Chain Reaction, Mutagenesis, Sequence Deletion, Mice, DNA Breaks, Double-Stranded, 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.
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