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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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.
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.
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.
(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.
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.
(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).
“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 Figures 1–7 (PDF 1632 kb)
Flow cytometry results and gRNA sequences. (XLSX 8 kb)
Automatic annotation of all PigA alleles in the study (XLSX 35 kb)
Detailed description of PigA alleles recovered from single cell clones. (XLSX 15 kb)
Diagnostic PCRs at the PigA locus. (XLSX 5 kb)
Summary of PCR genotyping experiments (XLSX 7 kb)
PCR primers. (XLSX 7 kb)
PigA alleles (TXT 820 kb)
Barcodes (ZIP 1 kb)
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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