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High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

Abstract

CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

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Figure 1: Identification and characterization of SpCas9 variants bearing substitutions in residues that form non-specific DNA contacts.
Figure 2: Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to standard, non-repetitive sites.
Figure 3: Validation of SpCas9-HF1 specificity improvements by deep sequencing of off-target sites identified by GUIDE-seq.
Figure 4: Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to non-standard, repetitive sites.
Figure 5: Activities of high-fidelity derivatives of SpCas9-HF1 bearing additional substitutions.

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Primary accessions

Sequence Read Archive

Data deposits

Plasmids encoding the high-fidelity SpCas9, VQR, and VRQR variants described in this manuscript have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas). All sequencing data from this study is available through the NCBI Sequence Read Archive (SRA) under accession number SRP066862.

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Acknowledgements

B.P.K. is supported by a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. V.P. was supported by the Massachusetts General Hospital (MGH) Department of Pathology. S.Q.T. is supported by an MGH Tosteson and Fund for Medical Discovery Fellowship. J.K.J. is supported by a US National Institutes of Health (NIH) Director’s Pioneer Award (DP1 GM105378), NIH R01 GM107427, NIH R01 GM088040, and the Jim and Ann Orr MGH Research Scholar Award.

Author information

Authors and Affiliations

Authors

Contributions

B.P.K., V.P., and J.K.J. conceived of and designed experiments. B.P.K., V.P., and M.S.P. performed all experiments. N.T.N. contributed to GUIDE-seq library preparation. B.P.K., V.P., M.S.P., S.Q.T., and Z.Z. analysed the data. B.P.K., V.P., and J.K.J. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to J. Keith Joung.

Ethics declarations

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. A patent application has been filed for high-fidelity Cas9 variants.

Extended data figures and tables

Extended Data Figure 1 SpCas9 interaction with the sgRNA and target DNA.

a, Schematic illustrating the SpCas9–sgRNA complex, with base pairing between the sgRNA and target DNA. b, Structural representation of the SpCas9-sgRNA complex bound to the target DNA, from PDB accession code 4UN3 (ref. 29). The four residues that form hydrogen bond contacts to the target-strand DNA backbone are highlighted in blue, the HNH domain is hidden for visualization purposes.

Extended Data Figure 2 On-target activities of high-fidelity SpCas9 variants.

a, b, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 (a) and SpCas9-HF1-derivative variants (b) in human cells. SpCas9-HF1 contains N497A, R661A, Q695A, and Q926A substitutions; HF2 = HF1 + D1135E; HF3 = HF1 + L169A; HF4 = HF1 + Y450A. Error bars represent s.e.m. for n = 3; mean level of background EGFP loss represented by the red dashed line.

Extended Data Figure 3 On-target activity comparisons of wild-type and SpCas9-HF1 with various sgRNAs used for GUIDE-seq experiments.

a, c, Mean GUIDE-seq tag integration at the intended on-target site for GUIDE-seq experiments shown in Figs 2a and Extended Data Fig. 5 (a and c, respectively), quantified by restriction-fragment length polymorphism assay. Error bars represent s.e.m. for n = 3. b, d, Mean percent modification at the intended on-target site for GUIDE-seq experiments shown in Fig. 2a and Extended Data Fig. 5 (b and d, respectively), detected by T7 endonuclease I assay. Error bars represent s.e.m. for n = 3.

Extended Data Figure 4 Positional summary of off-target sites identified by GUIDE-seq.

a, b, Heat maps derived from GUIDE-seq data with sgRNAs targeting non-repetitive (a), or repetitive or homopolymeric sites (b) in the genome are shown. Base frequencies in the set of all potential genomic off-target sites (weighted equally) with NGG PAMs and five or fewer mutations for each sgRNA are shown on the left. Summaries of off-target sites identified by GUIDE-seq for wild-type SpCas9 and SpCas9-HF1 (both weighted by read count) are shown on the right. Yellow box outlines denote on-target bases at each position. Positions (20–1) are shown below the heat maps, with 1 being the most PAM-proximal position. Note the presence of mismatches that would be expected to create potential wobble interactions (G→A or T→C) at certain positions among the off-target sites induced by wild-type SpCas9 and that SpCas9-HF1 appears to reduce off-target activity without any obvious positional bias.

Extended Data Figure 5 Genome-wide cleavage specificity of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to non-standard, repetitive sites.

a, GUIDE-seq profiles of wild-type SpCas9 and SpCas9-HF1 using two sgRNAs known to cleave large numbers of off-target sites4,8. GUIDE-seq read counts represent a measure of cleavage efficiency at a given site. Mismatched positions within the spacer or PAM are highlighted in colour red circles indicate off-target sites likely to have the indicated bulge12 at the sgRNA–DNA interface, blue circles indicate sites that may have an alternative gapped alignment relative to the one shown (see Extended Data Fig. 6). Off-target sites marked with red circles are not included in the counts of Fig. 4b, sites marked with blue circles are counted with the number of mismatches in the non-gapped alignment for Fig. 4b.

Extended Data Figure 6 Potential alternate alignments for VEGFA site 2 off-target sites.

Ten VEGFA site 2 off-target sites identified by GUIDE-seq (left) that may potentially be recognized as off-target sites with single nucleotide gaps12 (right), aligned using Geneious45 version 8.1.6 (http://www.geneious.com).

Extended Data Figure 7 Activities of wild-type SpCas9 and SpCas9-HF1 with truncated or 5′ mismatched sgRNAs14.

a, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 using full-length or truncated sgRNAs. b, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 using sgRNAs that encode a matched 5′ non-G nucleotide or an intentionally mismatched 5′ G nucleotide. For both panels, error bars represent s.e.m. for n = 3, and the mean level of background EGFP loss observed in control experiments is represented by the red dashed line.

Extended Data Figure 8 Altering the PAM recognition specificity of SpCas9-HF1.

a, Comparison of the mean per cent modification of on-target endogenous human sites by the SpCas9-VQR variant (ref. 15) and an improved SpCas9-VRQR variant using 8 sgRNAs, quantified by T7 endonuclease I assay. Both variants are engineered to recognize an NGAN PAM. Error bars represent s.e.m. for n = 3. b, On-target EGFP disruption activities of SpCas9-VQR and SpCas9-VRQR compared to their -HF1 counterparts using eight sgRNAs. Error bars represent s.e.m. for n = 3; mean level of background EGFP loss in negative controls represented by the red dashed line. c, Comparison of the mean on-target per cent modification by SpCas9-VQR and SpCas9-VRQR compared to their -HF1 variants at eight endogenous human gene sites, quantified by T7 endonuclease I assay. Error bars represent s.e.m. for n = 3; ND, not detectable. d, Summary of the fold-change in on-target activity when using SpCas9-VQR or SpCas9-VRQR compared to their corresponding -HF1 variants (from b and c). The median and interquartile range are shown, the interval showing greater than 70% of wild-type activity is highlighted in green.

Extended Data Figure 9 Titrations of wild-type SpCas9 and SpCas9-HF1 expression plasmid amounts.

Human cell EGFP disruption activities from transfections with varying amounts of wild-type and SpCas9-HF1 expression plasmids. For all transfections, the amount of sgRNA-containing plasmid was fixed at 250 ng. Two sgRNAs targeting different sites were used; Error bars represent s.e.m. for n = 3; mean level of background EGFP loss in negative controls is represented by the red dashed line.

Extended Data Table 1 Summary of potential mismatched sites in the reference human genome for the ten sgRNAs examined by GUIDE-seq

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, additional references and Supplementary Sequences (a subset of plasmids used in this study). (PDF 297 kb)

Supplementary Table 1

This table contains the sgRNA targets. (XLSX 17 kb)

Supplementary Table 2

This table contains the oligonucleotides used in this study. (XLSX 11 kb)

Supplementary Table 3

This table, which has multiply tabs, contains the p-values for data from Figures 1 and 5. (XLSX 66 kb)

Supplementary Table 4

This table, which has multiply tabs, contains the summary of GUIDE-seq data. (XLSX 112 kb)

Supplementary Table 5

This table, which has multiply tabs, contains the targeted deep sequencing amplicons and data. (XLSX 82 kb)

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Kleinstiver, B., Pattanayak, V., Prew, M. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016). https://doi.org/10.1038/nature16526

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