Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

  • An Erratum to this article was published on 01 October 2018

Abstract

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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Figure 1: Frequency of PigA loss upon editing with exonic and intronic gRNAs in mouse ES cells.
Figure 2: Analysis of the PigA locus edited with selected gRNAs.
Figure 3: Analysis of Cas9 editing at the autosomal Cd9 locus in mouse ES cells.
Figure 4: Frequency of PIGA loss upon editing with exonic and intronic gRNAs and structure of the recovered alleles in human RPE1 cells.

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Change history

  • 31 July 2018

    In the version of this article initially published online, four figure citations were incorrect on p.2: left-hand column, after "complex rearrangements," "Supplementary Fig. 2a,b" should have been "Fig. 2a,b"; right-hand column, in three places, the citation for "Supplementary Fig. 3..." should have been for "Supplementary Fig. 2." The errors have been corrected for the print, PDF and HTML versions of this article.

  • 01 October 2018

    Nat. Biotechnol. 10.1038/nbt.4192; corrected online 31 July 2018 In the version of this article initially published online, four figure citations were incorrect on p.2: left-hand column, after “complex rearrangements,” “Supplementary Fig. 2a,b” should have been “Fig. 2a,b”; right-hand column, in three places, the citation for “Supplementary Fig.

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Acknowledgements

We wish to thank M. Friedrich for sharing his gRNA expression construct and technical advice, E. Metzakopian for technical advice and critical reading of the manuscript, G. Rutledge for critical reading of the early manuscript, A. Ferguson-Smith for the CAST/B6 hybrid ES cells, P. Liu and X. Gao for mCherry/GFP reporter cells, S. Jackson's group for the Cas9-expressing RPE1 cell line and the Cytometry Core Facility for assistance with cell sorting. This work was supported by the Wellcome Trust Grant number 098051.

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Contributions

M.K. performed most of the experiments and analyzed the data. K.T. performed the primary cell work. A.B. supervised the project. All authors contributed to writing of the manuscript.

Corresponding author

Correspondence to Allan Bradley.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Frequency of PigA loss upon editing with RNP in mouse ES cells and structure of the recovered alleles.

(A) Frequency of PigA loss caused by Cas9 with intronic and exonic gRNAs. Each experiment was conducted in biological triplicate and technical duplicate. (B) Recovered alleles from the “Neon electroporation” experiment. Display conventions as in Fig. 4. Alleles are sorted by total length. See Table S2 for detailed descriptions.

Supplementary Figure 2 Examples of insertion alleles at the PigA locus.

Panels, A, B and C are illustrations of different alleles. The bottom of each panel is a representation of the PigA reference allele around exon 2, the top of each panel shows the structure of the sequenced allele. Black horizontal line – direct reference match, orange bar – inversion, blue bar – insertion from another part of the genome, black arrowhead – gRNA target site, violet bar – duplication, black vertical bar – small indel within the insertion. Grey and orange shadows represent, respectively, direct and inverted match between the reference and the sequenced allele. Lack of shadow at the reference locus represents a deletion in the sequenced allele.

Supplementary Figure 3 Structure of recovered alleles from PigA-deficient, mouse ES cell clones.

(A) Positions of primer pairs used to screen PigA-deficient clones. In grey, primers used for diagnostic PCRs on alleles that could not be recovered. Other display conventions as in Fig. 3. (B-D) Recovered alleles. (B) 5' intronic guide, (C) 3' intronic guide, (D) exonic guide. Display conventions as in Fig. 4. Alleles are sorted by total length.

Supplementary Figure 4 Analysis of the diversity of editing outcomes at PigA locus.

PigA locus was edited using 5’ intronic guide (#15) in biological quadruplicate. Duplicate PCR reactions spanning the cut site performed on DNA extracted from the bulk of PigA negative cells and resolved on an agarose gel (product size 3500bp, 1F/1R primer pair, Fig. S2A). (A) Experimental considerations. (B) Deletion fingerprints. Cell line: JM8 – the original mouse embryonic stem cell line (transfected with gRNA and Cas9 PiggyBac vectors); CAS – subclone of JM8 expressing Cas9 from a single-copy lentiviral transgene (transfected with only gRNA PiggyBac vector). Ladder scale is in kilobases.

Supplementary Figure 5 Genotyping of GFP-deficient progenitor cells.

(A) Positions of primer pairs and the gRNA. Display conventions as in Fig. 3A. (B) Agarose gel pictures. Asterisks indicate clones with at least one deletion allele. Red asterisks indicate eight clones, whose deletion alleles were verified by Sanger sequencing (Fig. S6A). Ladder scale is in kilobases.

Supplementary Figure 6 Structure of recovered deletion alleles from GFP-deficient clones and gating strategy for Cd9 cell sorting.

(A) Display conventions as in Fig. S3. (B) Cells were edited with various intronic and exonic gRNAs, sorted for “low”, “medium” and “high” classes and single cell cloned (Table S5; sorting was performed twice for guides #1 and #86 and once for guides #35 and #80; flow cytometric analysis was replicated independently 4-7 times, see also Table S1).

Supplementary Figure 7 Overlap between unique PigA alleles derived using different methods.

“Single cell” and “PacBio” refer to alleles shown in Fig. S2B-D and Fig. 2A. “Plasmid” alleles were derived in the same experiment by Sanger sequencing of 3.5kb long PCR fragments cloned into plasmid vectors (primer pair 1F/1R, Fig. S2A). “RNP” alleles were derived by single cell cloning in an independent experiment only using guide #15 (Fig. S1A “Electroporation exp2”; Fig. S1B).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1632 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Table 1

Flow cytometry results and gRNA sequences. (XLSX 8 kb)

Supplementary Table 2

Automatic annotation of all PigA alleles in the study (XLSX 35 kb)

Supplementary Table 3

Detailed description of PigA alleles recovered from single cell clones. (XLSX 15 kb)

Supplementary Table 4

Diagnostic PCRs at the PigA locus. (XLSX 5 kb)

Supplementary Table 5

Summary of PCR genotyping experiments (XLSX 7 kb)

Supplementary Table 6

PCR primers. (XLSX 7 kb)

Supplementary Data 1

PigA alleles (TXT 820 kb)

Supplementary Data 2

Barcodes (ZIP 1 kb)

Supplementary Note

Supplementary Note 1 (PDF 169 kb)

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Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765–771 (2018). https://doi.org/10.1038/nbt.4192

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