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Mapping the genomic landscape of CRISPR–Cas9 cleavage

Abstract

RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

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Figure 1: SITE-Seq workflow.
Figure 2: SITE-Seq target sites recovered with 0.25–1,024 nM Cas9.
Figure 3: SITE-Seq predicts cell-based activity.
Figure 4: Off-target editing varies with sgRNP delivery method, duration of treatment and cell type.
Figure 5: Comparison of SITE-Seq with other off-target analysis methods.

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Acknowledgements

The authors thank R. Haurwitz, S. Sternberg, B. McClung, and the members of Caribou Biosciences for providing helpful comments on the manuscript.

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Authors

Contributions

J.K.Y., V.L., S.D., M.C., A.P.M. and P.D.D. designed critical proof-of-concept experiments to initially develop the SITE-Seq protocol. P.C. performed all SITE-Seq experiments in this study; B.N.J. performed amplicon digestion experiments; M.S.T. and L.M.B. performed sequencing for cell-based validation experiments. P.C., E.M.S., B.V., E.G. and E.L. performed cell-based validation experiments. S.G., A.M.L. and L.S.E. generated sgRNA and other reagents. P.C., C.K.F., P.D.D., M.M.C., D.C., M.C., B.N.J., A.H.S., J.K.Y. and A.P.M. analyzed the data. P.C., C.K.F., P.D.D., M.C., J.K.Y. and A.P.M. wrote the manuscript.

Corresponding authors

Correspondence to Joshua K Young or Andrew P May.

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Competing interests

A.P.M., P.D.D., P.C., C.K.F., M.S.T., M.M.C., S.G., B.N.J., E.L., E.M.S., B.V., E.L., L.M.B., A.M.L., L.S.E., A.H.S., and D.C. are current or former employees of Caribou Biosciences, Inc., a company that develops and commercializes genome engineering technologies; and such individuals may own shares or stock options in Caribou Biosciences, Inc. J.K.Y., V.L., and S.D. are employees of DuPont Pioneer. Patent applications have been filed describing this methodology; see, for example, PCT publication no. WO2014/164466, published October 9, 2014.

Integrated supplementary information

Supplementary Figure 1 Sequence logos resulting from SITE-Seq target sites recovered with 0.25 nM – 1,024 nM sgRNP.

The total number of SITE-Seq target sites recovered at each sgRNP concentration is displayed (upper left corner) with each logo. On-target sequences are directly below the gene name. The concentration of sgRNP used in SITE-Seq is shown to the right.

Supplementary Figure 2 SITE-Seq target sites with higher biochemical cleavage sensitivities are more likely to show cell-based editing.

(a) For each sgRNA, the fraction of SITE-Seq target sites showing cell-based editing as a function of SITE-Seq digestion conditions. (b) Across all sites examined in cells, the fraction of SITE-Seq target sites showing cell-based editing as a function of SITE-Seq digestion conditions.

Supplementary Figure 3 DNase-I hypersensitivity does not correlate with on- or off-target activity.

DNase-I hypersensitivity does not correlate with on- or off-target activity. (a) Scatterplots of indel frequencies at on- and -off-target sites as a function of enrichment scores for DNase-I hypersensitivity density (generated by F-Seq). sgRNAs targeting AAVS1, VEGFA, or FANCF were transfected into HEK293 cells stably expressing Cas9-GFP and on- and off-target editing was measured three days later. (b) Scatterplots of indel frequencies at SITE-Seq target sites directly targeted with sgRNAs as a function of enrichment scores for DNase-I hypersensitivity density (generated by F-Seq). 387 sgRNAs were transfected into HEK293 cells stably expressing Cas9-GFP and on-target editing at each SITE-Seq target site was measured two days later.

Supplementary Figure 4 SITE-Seq mediated sgRNA selection.

(a) The EGFR gene was targeted with 83 sgRNAs tiling across six exons. Shown is the full EGFR locus as well as the first six exons and their corresponding guides (grey guides target sense strand and red guides target anti-sense strand). (b) Overlap between SITE-Seq target sites recovered with 4 nM positive control sgRNPs and cellular off-targets observed after transient transfection of pre-assembled sgRNP. (c) Dot plots show coupling between EGFR protein knockdown and recovery of SITE-Seq target sites. sgRNAs with high on-target activity (>70% EGFR knockdown) and less than 30 SITE-Seq target sites are shown as either orange (if at least one SITE-Seq target site is found in an exon) or green (if no SITE-Seq target site is found in an exon).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 792 kb)

Supplementary Table 1–9

SITE-Seq target sites recovered (XLSX 1207 kb)

Supplementary Table 10

SITE-Seq target sites recovered with VEGFA sgRNP that were segregated by MEME into a second motif. (XLSX 114 kb)

Supplementary Table 11

Biochemical cleavage of amplicons containing SITE-Seq target sites, as a function of sgRNP concentration. (XLSX 14 kb)

Supplementary Table 12–19

SITE-Seq target sites examined in cell-based validation. (XLSX 208 kb)

Supplementary Table 20–22

SITE-Seq target sites examined in cell-based validation with a panel of delivery methods. (XLSX 71 kb)

Supplementary Table 23

SITE-Seq with high sequencing coverage. (XLSX 62 kb)

Supplementary Table 24

Comparing SITE-Seq data with in silico approaches (XLSX 9 kb)

Supplementary Table 25-26

Oligonucleotides used in SITE-Seq. (XLSX 11 kb)

Supplementary Protocol

SITE-Seq Supplementary Protocol (PDF 1253 kb)

Supplementary Software

SITE-Seq feature calling function (TXT 8 kb)

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Cameron, P., Fuller, C., Donohoue, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat Methods 14, 600–606 (2017). https://doi.org/10.1038/nmeth.4284

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