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. 2016 Jan 28;529(7587):490-5.
doi: 10.1038/nature16526. Epub 2016 Jan 6.

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

Benjamin P Kleinstiver  1   2 , Vikram Pattanayak  1   2 , Michelle S Prew  1 , Shengdar Q Tsai  1   2 , Nhu T Nguyen  1 , Zongli Zheng  3 , J Keith Joung  1   2
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High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects

Benjamin P Kleinstiver et al. Nature. .
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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.

Conflict of interest statement

Competing financial 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.

Figures

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: 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 and b, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 (panel a) and SpCas9-HF1-derivative variants (panel b) in human cells. SpCas9-HF1 contains N497A, R661A, Q695, 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 and 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 (panels a and c, respectively), quantified by restriction fragment length polymorphism assay. Error bars represent s.e.m. for n = 3. b and d, Mean percent modification at the intended on-target site for GUIDE-seq experiments shown in Figs. 2a and Extended Data Fig. 5 (panels b and d, respectively), detected by T7E1 assay. Error bars represent s.e.m. for n = 3.
Extended Data Figure 4. Positional summary of off-target sites identified by GUIDE-seq
Heat maps derived from GUIDE-seq data with sgRNAs targeting a, non-repetitive, or b, repetitive or homopolymeric sites 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 improve off-target sites 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 sites, . GUIDE-seq read counts represent a measure of cleavage efficiency at a given site; mismatched positions within the spacer or PAM are highlighted in color; red circles indicate sites likely to have the indicated bulge 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 gaps (right), aligned using Geneious version 8.1.6 (http://www.geneious.com).
Extended Data Figure 7. Activities of wild-type SpCas9 and SpCas9-HF1 with truncated and 5’ mismatched sgRNAs
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 percent 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 T7E1 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 percent modification by SpCas9-VQR and SpCas9-VRQR compared to their -HF1 variants at eight endogenous human gene sites, quantified by T7E1 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 panels 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.
Figure 1. Identification and characterization of SpCas9 variants bearing substitutions in residues that form non-specific DNA contacts
a, Schematic depicting wild-type SpCas9 interactions with the target DNA:sgRNA duplex, based on PDB 4OO8 and 4UN3 (adapted from refs. and , respectively). b, Characterization of SpCas9 variants that contain alanine substitutions in positions that form hydrogen bonds with the DNA backbone. Wild-type SpCas9 and variants were assessed using the human cell EGFP disruption assay when programmed with a perfectly matched sgRNA or partially mismatched sgRNAs. Error bars represent s.e.m. for n = 3; mean level of background EGFP loss represented by red dashed line. c, On-target activities of wild-type SpCas9 and SpCas9-HF1 across 13 endogenous sites measured by T7E1 assay. Error bars represent s.e.m. for n = 3. d, Ratio of on-target activity of SpCas9-HF1 to wild-type SpCas9. The median and interquartile range are shown; the interval with >70% of wild-type activity is highlighted in green.
Figure 2. Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to standard, non-repetitive sites
a, Off-target cleavage sites of wild-type SpCas9 and SpCas9-HF1 with eight sgRNAs targeted to endogenous human genes, as determined by GUIDE-seq. Read counts represent a measure of cleavage frequency at a given site; mismatched positions within the spacer or PAM are highlighted in color. b, Summary of the total number of genome-wide off-target sites identified by GUIDE-seq for wild-type SpCas9 and SpCas9-HF1 with the sgRNAs used in panel a. c, Off-target sites identified for wild-type SpCas9 and SpCas9-HF1 for the eight sgRNAs, binned according to the total number of mismatches (in the protospacer and PAM) relative to the on-target site.
Figure 3. Validation of SpCas9-HF1 specificity improvements by deep sequencing of off-target sites identified by GUIDE-seq
a, Mean on-target percent modification for wild-type SpCas9 and SpCas9-HF1 with six sgRNAs from Fig. 2. Error bars represent s.e.m. for n = 3. b, Percent modification of on-target and GUIDE-seq detected off-target sites with indel mutations. Triplicate experiments are plotted for wild-type SpCas9, SpCas9-HF1, and a negative control; off-target sites are numbered as indicated in Fig. 2a. Filled circles below the x-axis represent replicates for which no insertion or deletion mutations were observed (Supplementary Table 4). Hypothesis testing using a one-sided Fisher exact test with pooled read counts found significant differences (p < 0.05 after adjusting for multiple comparisons using the Benjamini-Hochberg method) for comparisons between SpCas9-HF1 and the control condition only at EMX1-1 off-target 1 and FANCF-3 off-target 1. Significant differences were also found between wild-type SpCas9 and SpCas9-HF1 at all off-target sites, and between wild-type SpCas9 and the control condition at all off-target sites except RUNX1-1 off-target 2. c, Scatter plot of the correlation between GUIDE-seq read counts (from Fig. 2a) and mean percent modification determined by deep sequencing at on- and off-target cleavage sites with wild-type SpCas9.
Figure 4. Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to non-standard, repetitive sites
a, Summary of the total number of genome-wide off-target cleavage sites identified by GUIDE-seq for wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to VEGFA sites 2 and 3. b, Off-target sites identified for wild-type SpCas9 or SpCas9-HF1 with sgRNAs targeted VEGFA sites 2 and 3 binned according to the total number of mismatches (within the protospacer and PAM) relative to the on-target site.
Figure 5. Activities of high-fidelity derivatives of SpCas9-HF1 bearing additional substitutions
a, Summary of the on-target EGFP disruption activities of various SpCas9-HF variants compared to wild-type SpCas9 (from the data in Extended Data Fig. 2b). SpCas9-HF1 contains N497A, R661A, Q695, and Q926A substitutions; HF2 = HF1 + D1135E; HF3 = HF1 + L169A; HF4 = HF1 + Y450A. The median and interquartile range are shown; the interval showing >70% of wild-type activity is highlighted in green. b, Mean percent modification by SpCas9 and HF variants at the FANCF site 2 and VEGFA site 3 on-target sites, as well as off-target sites from Figs. 2a and Extended Data Fig. 5 resistant to the effects of SpCas9-HF1. Percent modification determined by T7E1 assay; background indel percentages were subtracted for all experiments; error bars represent s.e.m. for n = 3. c, Specificity ratios of wild-type SpCas9 and HF variants with the FANCF site 2 or VEGFA site 3 sgRNAs, plotted as the ratio of on-target to off-target activity (from panel b).
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