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Cited by 117CRISPR and Genetic EngineeringDNA Repair MechanismsRNA regulation and disease

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

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Kosicki, Michael, Allen, Felicity, Steward, Frances, Tomberg, Kärt, Pan, Yangyang, Bradley, Allan·2022·Nature Communications
DOI 10.1038/s41467-022-30480-8PMC PMC9197861

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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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Fig. 1 End joining pathways divergently control the frequency of large deletions caused by Cas9 in mouse ES cells. A Experimental design. Library of Cas9-positive NGS-validated clones deficient for DNA damage repair genes was transfected with individual gRNA-expressing constructs and selected for stable integration. Expression of target genes was measured by flow cytometry, revealing frequency of large deletions (using intronic gRNA #15) or overall mutagenesis (using gRNAs #48U, #48 and #148). Frequency of small indels was established by targeted sequencing of short-range PCR products. B Frequency of large deletions caused by Cas9 with intronic gRNA in DNA damage deficient clones, measured by flow cytometry, expressed as a regressed z-score (see Methods). Only the initial clone in a series of clones deficient for the same gene is labeled on the x axis. N = 4 independent cell cultures. C Comparison of large deletion and mutagenesis indices (see Methods). Dashed line indicates best linear fit to control clones (in orange). Error bars are 2xSEM (N = 4). D Correlation between large deletion z-score (measured by flow cytometry) and the size or microhomology extent of small indels (measured by targeted sequencing). Each dot represents an average readout of an individual clone (N = 1–2 biologically independent cell cultures). Negative indel sizes indicate dominance of deletions.
Fig. 1 End joining pathways divergently control the frequency of large deletions caused by Cas9 in mouse ES cells. A Experimental design. Library of Cas9-positive NGS-validated clones deficient for DNA damage repair genes was transfected with individual gRNA-expressing constructs and selected for stable integration. Expression of target genes was measured by flow cytometry, revealing frequency of large deletions (using intronic gRNA #15) or overall mutagenesis (using gRNAs #48U, #48 and #148). Frequency of small indels was established by targeted sequencing of short-range PCR products. B Frequency of large deletions caused by Cas9 with intronic gRNA in DNA damage deficient clones, measured by flow cytometry, expressed as a regressed z-score (see Methods). Only the initial clone in a series of clones deficient for the same gene is labeled on the x axis. N = 4 independent cell cultures. C Comparison of large deletion and mutagenesis indices (see Methods). Dashed line indicates best linear fit to control clones (in orange). Error bars are 2xSEM (N = 4). D Correlation between large deletion z-score (measured by flow cytometry) and the size or microhomology extent of small indels (measured by targeted sequencing). Each dot represents an average readout of an individual clone (N = 1–2 biologically independent cell cultures). Negative indel sizes indicate dominance of deletions.CC BY · PubMed Central
Fig. 2 Core end joining genes influence indel profiles globally. A Indel profiles in combined 12 control samples. B Relationships between cell clones based on their indel profiles. Clones significantly different from controls in both replicates are labeled (FDR-corrected p < 0.01 from a chi-squared distribution, see Methods). The arrangement of non-significant clones is in Supplementary Fig. 3C. C Correlation between mean indel size and microhomology. D Relative frequencies of indel sizes compared to controls in deficient clones with a significant impact on all three gRNAs. Indel profiles of other clones with significant impact are in Supplementary Fig. 6. Y axis is truncated at −15% and +15%. In panels (A) and (D), indel frequencies are aggregated by combined size. Negative numbers represent deletions and positive ones represent insertions. The leftmost and rightmost bars (−30 and 10) combine all larger deletions and insertions, respectively. Biological replicates (N = 2) were averaged for clarity. All rows in panels (B) and (C) relate to the same gRNAs as in panel (A). In panels (B) and (C), controls are in orange and samples are in blue.
Fig. 2 Core end joining genes influence indel profiles globally. A Indel profiles in combined 12 control samples. B Relationships between cell clones based on their indel profiles. Clones significantly different from controls in both replicates are labeled (FDR-corrected p < 0.01 from a chi-squared distribution, see Methods). The arrangement of non-significant clones is in Supplementary Fig. 3C. C Correlation between mean indel size and microhomology. D Relative frequencies of indel sizes compared to controls in deficient clones with a significant impact on all three gRNAs. Indel profiles of other clones with significant impact are in Supplementary Fig. 6. Y axis is truncated at −15% and +15%. In panels (A) and (D), indel frequencies are aggregated by combined size. Negative numbers represent deletions and positive ones represent insertions. The leftmost and rightmost bars (−30 and 10) combine all larger deletions and insertions, respectively. Biological replicates (N = 2) were averaged for clarity. All rows in panels (B) and (C) relate to the same gRNAs as in panel (A). In panels (B) and (C), controls are in orange and samples are in blue.CC BY · PubMed Central
Fig. 3 Deficiencies in specialized DNA damage repair genes influence indel profiles in a locus-specific manner. A Indel profile divergence between controls and selected clones. Blue bars highlight clone/gRNA combinations that were significantly affected (FDR-corrected p < 0.01 from a chi-squared distribution, see Methods). B Change in frequency of individual indels relative to controls. 'D' = deletion, 'I' = insertion, 'M' = microhomology (see Methods). X axis indicates the frequency in control clones. Y axis indicates relative change in indel frequency in a given clone relative to control clones. Complete loss of the indel is at −100%, while 100% indicates doubled frequency. The axis is truncated there for display clarity. Only indels present at 0.3% frequency or higher in control clones are shown. Dashed lines indicate absolute change of 0.1% and 1% respectively, color gradations highlight this change. Biological replicates (N = 2) were averaged for clarity. All columns relate to the same gRNAs as in panel (A).
Fig. 3 Deficiencies in specialized DNA damage repair genes influence indel profiles in a locus-specific manner. A Indel profile divergence between controls and selected clones. Blue bars highlight clone/gRNA combinations that were significantly affected (FDR-corrected p < 0.01 from a chi-squared distribution, see Methods). B Change in frequency of individual indels relative to controls. 'D' = deletion, 'I' = insertion, 'M' = microhomology (see Methods). X axis indicates the frequency in control clones. Y axis indicates relative change in indel frequency in a given clone relative to control clones. Complete loss of the indel is at −100%, while 100% indicates doubled frequency. The axis is truncated there for display clarity. Only indels present at 0.3% frequency or higher in control clones are shown. Dashed lines indicate absolute change of 0.1% and 1% respectively, color gradations highlight this change. Biological replicates (N = 2) were averaged for clarity. All columns relate to the same gRNAs as in panel (A).CC BY · PubMed Central
Fig. 4 Efficiency of mutagenesis affects DNA repair outcomes. A Relationship between flow cytometry samples. gRNA #48U is a weaker version of #48. N = 4–6 biologically independent replicates. B Relationship between clones based on their indel profiles, analogous to Fig. 2B. Only non-significant clones are shown for clarity. Biological replicates (N = 2) were averaged. C Difference in indel frequency between the regular gRNA #48 and its less active counterpart #48U. Same display conventions as in Fig. 2. D Relationship between clones based on their indel profiles, analogous to Fig. 2B. Each clone is colored by the frequency of mutagenesis assayed by flow cytometry on day 14 using gRNA #48U, a proxy for Cas9 activity. For clarity, only non-significant clones are depicted.
Fig. 4 Efficiency of mutagenesis affects DNA repair outcomes. A Relationship between flow cytometry samples. gRNA #48U is a weaker version of #48. N = 4–6 biologically independent replicates. B Relationship between clones based on their indel profiles, analogous to Fig. 2B. Only non-significant clones are shown for clarity. Biological replicates (N = 2) were averaged. C Difference in indel frequency between the regular gRNA #48 and its less active counterpart #48U. Same display conventions as in Fig. 2. D Relationship between clones based on their indel profiles, analogous to Fig. 2B. Each clone is colored by the frequency of mutagenesis assayed by flow cytometry on day 14 using gRNA #48U, a proxy for Cas9 activity. For clarity, only non-significant clones are depicted.CC BY · PubMed Central

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Metadata

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
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-06-14
Volume
13
Issue
1
Pages
3422-3422
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-30480-8
PMC
PMC9197861
OpenAlex
W4282823317
Cited by
115
References
69
Citations by year
2026: 27 · 2025: 28 · 2024: 26 · 2023: 26 · 2022: 4
FWCI
7.02
Type
journal article
Language
en
Open access
Licence
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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