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Cited by 216CRISPR and Genetic EngineeringDNA Repair MechanismsRNA regulation and disease

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

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Chen, Wei, McKenna, Aaron, Schreiber, Jacob, Haeussler, Maximilian, Yin, Yi, Agarwal, Vikram, Noble, William Stafford, Shendure, Jay·2019·Nucleic Acids Research
DOI 10.1093/nar/gkz487PMC PMC6735782

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Abstract

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.

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  • gRNA-Cas9 nuclease-induced DSBs repaired by NHEJ resulted in 1-10 bp deletions or 1 bp insertions in as many as 90% of cells.

    “The gRNA–Cas9 nuclease complex directed double-stranded breaks (DSBs) at target sites, which after repair by error-prone non-homologous end-joining (NHEJ)196,197, resulted in 1–10 bp deletions or 1 bp insertions (‘indels’) in as many as 90% of cells192,194 (Figure 2a).”

    From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [197]

    The Results section, “Development of a massively parallel strategy to profile NHEJ-mediated genome edits” (page unavailable), reports that only 9.8% of measured UMIs were wild type, deletions were predominantly small, and most insertions were 1 bp. The “Extensive use of microhomology in NHEJ-mediated deletions” section describes common deletion lengths within 1–10 bp. But the paper does not establish that 90% of cells had specifically 1–10 bp deletions or 1 bp insertions: its approximately 90% figure concerns edited UMIs, not cells or that restricted set of outcomes. (LLM, conf=0.87)

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Sorted by citations

Authors
Chen, W., McKenna, A., Schreiber, J., Haeussler, M., Yin, Y., Agarwal, V., Noble, W. S., & Shendure, J.
Affiliations
  • University of Washington (US)
  • University of California, Santa Cruz (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Shendure, Jay
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2019-06-05
Volume
47
Issue
15
Pages
7989-8003
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkz487
PMC
PMC6735782
OpenAlex
W2970098032
Cited by
216
References
9
Citations by year
2026: 26 · 2025: 25 · 2024: 39 · 2023: 23 · 2022: 34 · 2021: 32 · 2020: 31 · 2019: 6
FWCI
8.52
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
  • Paul G. Allen Frontiers Foundation
  • NIH (R01HG009136, UM1HG009408, 1K99HG010152-01)
  • National Human Genome Research Institute (4U41HG002371)
  • St. Baldricks Foundation (427053)
  • Chan-Zuckerberg Initiative (SVCF 2018-182809)
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
CRISPR/Cas9, non-homologous end joining, DNA double-strand break repair, microhomology, indels, genome-wide screen
MeSH terms
Gene Editing, Base Sequence, Humans, Models, Genetic, Mutation, Genome, Human, Sequence Deletion, DNA Cleavage, DNA Breaks, Double-Stranded, DNA End-Joining Repair, CRISPR-Cas Systems

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