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. 2017 Oct 19;550(7676):407-410.
doi: 10.1038/nature24268. Epub 2017 Sep 20.

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

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Free PMC article

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

Janice S Chen et al. Nature. .
Free PMC article

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS (to be included in online version only)

J.K.J. has financial interests in Beacon Genomics, Beam Therapeutics, Editas Medicine, Pairwise Plants, 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. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, and Intellia Therapeutics; a scientific advisor to Caribou, Intellia, eFFECTOR Therapeutics and Driver; and executive director of the Innovative Genomics Institute at UC Berkeley and UCSF. S.H.S. is an employee of Caribou Biosciences, Inc. S.H.S., J.S.C. and J.A.D. are inventors on a patent application entitled “Reporter Cas9 variants and methods of use thereof” (PCT/US2016/036754), filed by The Regents of the University of California. B.P.K. and J.K.J. are inventors on a patent application entitled “Engineered CRISPR-Cas9 nucleases” (US 15/060,424), filed by The General Hospital Corporation. J.S.C, Y.S.D., B.P.K, A.Y., J.K.J. and J.A.D. have filed a patent application related to this work through The General Hospital Corporation and The Regents of the University of California.

Figures

Extended Data Figure 1
Extended Data Figure 1. Dually-labeled SpCas9 variants are fully functional for DNA cleavage
a, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of unlabeled Cas9 variants. b, SDS-PAGE analysis of Cy3/Cy5-labeled Cas9 variants. The gel was scanned for Cy3/Cy5 fluorescence (middle, bottom) before staining with Coomassie blue (top). c–f, DNA cleavage time courses of Cas9 FRET constructs and their dually-labeled counterparts for c, WT SpCas9, d, SpCas9-HF1, e, eSpCas9(1.1) and f, HypaCas9. For panels a–f, experiments were repeated three independent times with similar results.
Extended Data Figure 2
Extended Data Figure 2. HNH domain in eSpCas9 variants still populate the docked state in the presence of PAM-distal mismatches
a, Quantification of DNA cleavage time courses comparing WT SpCas9, SpCas9-HF and eSpCas9(1.1) variants with perfect and PAM-distal mismatched targets. b, Dissociation constants comparing WT SpCas9, SpCas9-HF and eSpCas9(1.1) variants with perfect and PAM-distal mismatched targets, as measured by electrophoretic mobility shift assays. For panels a–b, mean and s.d. shown; n = 3 independent experiments (overlaid as white circles in panel b). c–d, smFRET histograms for c, SpCas9-K855A and d, SpCas9-N497A/R661A/Q695A. For panels c and d, black curves represent a fit to multiple Gaussian peaks. e, Schematic of SpCas9 domain structure with color coding for separate domains. f, Vector map of global SpCas9 conformational changes from the sgRNA- (PDB ID: 4ZT0) to dsDNA-bound structures (PDB ID: 5F9R), domains colored as in panel e.
Extended Data Figure 3
Extended Data Figure 3. Kinetic analysis of transitions between active and inactive states of the HNH domain
a, Representative time traces (top), transition density plots (TDPs, middle) and rates of the transitions in TDPs (bottom) for SpCas9-HF1 with on-target DNA (left), eSpCas9(1.1) with on-target DNA (middle) and eSpCas9(1.1) with 20-20 bp mm DNA (right); mean and s.e.m. shown; n = 107, 24, and 74 individual molecules, respectively. The percentage of molecules that show at least one such transitions was 36%, 7% and 29% for SpCas9-HF1 with on-target, eSpCas9(1.1) with on-target and eSpCas9(1.1) with 20-20 bp mm DNA, respectively. Kinetics analysis of other cases (SpCas9-HF1 and eSpCas9(1.1) bound to other off-target substrates, and HypaCas9 bound to on- and off-target substrates) is not shown, because the percentage of molecules that show at least one such transitions was less than 3%. b, Comparison of on-target transition rates for WT SpCas9, SpCas9-HF1, and eSpCas9(1.1); mean and s.e.m. shown; n = 51, 107 and 24 individual molecules, respectively. Transition rates for WT SpCas9 collected from ref. .
Extended Data Figure 4
Extended Data Figure 4. Nucleic acid sensing requires engagement with the REC3 domain and outward rotation of the REC2 domain
a, Schematic of SpCas9REC3 with FRET dyes at positions S701C and S960C, with HNH domain omitted for clarity. Inactive to active structures represent REC3 in the sgRNA-bound (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. b–c, smFRET histograms showing HNH conformational activation with black curves representing a fit to multiple Gaussian peaks for b, WT SpCas9REC3 and c, SpCas9-HF1REC3 bound to perfect and PAM-distal mismatched targets. The purple peak denotes the sgRNA-only bound state, while the red and green peaks represent two states of REC3 with conformational flexibility upon binding to DNA substrates. d, REC3 in vitro complementation assay with SpCas9ΔREC3 by measuring cleavage rate constants. e, On-target DNA binding assay in the presence or absence of the REC3 domain; mean and s.d. shown. f, REC3 in vitro complementation assay with SpCas9ΔREC3 by measuring HNH activation with (ratio)A values. g, (Ratio)A data with SpCas9REC2 and SpCas9HNH showing reciprocal FRET states with the indicated substrates. For panels d–g, mean and s.d. shown; n = 3 independent experiments (overlaid as white circles in panels d, f, and g). h, Schematic of SpCas9ΔREC3REC2 with FRET dyes at positions E60C and D273C, with the REC3 domain added in trans. Inactive to active structures represent REC2 in the sgRNA-bound (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. i, smFRET histograms measuring REC2 conformational states with SpCas9ΔREC3REC2 in the absence and presence of the REC3 domain when bound to an on-target substrate.
Extended Data Figure 5
Extended Data Figure 5. Identification of Cluster variants based on nucleic acid proximity and multiple sequence alignment of residues within Clusters 1–5
a, Schematic depicting interactions of WT SpCas9 residues within Clusters 1–5 with the RNA/DNA heteroduplex, based on PDB accession 5F9R (adapted from ref 9). b, Alignment of selected Cas9 orthologues using MAFFT and visualized in Geneious 10.0, with red boxes outlining residues mutated to alanine within each cluster variant.
Extended Data Figure 6
Extended Data Figure 6. Mutation clusters in the REC3 domain along the RNA/DNA heteroduplex demonstrate localized sensitivity to mismatches along the target sequence
a–b, Quantified DNA cleavage rates (dotted line indicates detection limit for kcleave set at 10 min−1) displayed as a a, heatmap and b, bar graph. c–d, Target DNA binding assay c, resolved by native polyacrylamide gel electrophoresis (PAGE) mobility shift assays; repeated three independent times with similar results and d, quantification with WT-normalized dissociation constants. For panels b and d, mean and s.d. shown; n = 3 independent experiments (overlaid as white circles).
Extended Data Figure 7
Extended Data Figure 7. On-target activities of altered specificity variants using a human cell EGFP disruption assay
a, Summary of EGFP disruption activities for SpCas9-HF1, eSpCas9(1.1), eSpCas9(1.1)-HF1 and Cluster variants ± Q926A with mean and s.e.m., where n = at least 3 biologically independent samples (overlaid as white circles). b, Summary of EGFP disruption activities for the series of Cluster 1 variants with each substituted residue restored to the canonical amino acid; mean and s.e.m. where n = at least 3 biologically independent samples (overlaid as white circles); WT, Cluster 1 (HypaCas9), and Cluster 1 + Q926A data from panel a is re-plotted for comparison. c, WT-normalized plot of data in panel b; error bars represent median and interquartile range for n = 12 biologically independent samples; the interval with >70% of WT activity is highlighted in light grey.
Extended Data Figure 8
Extended Data Figure 8. Activities and specificities of high-fidelity SpCas9 variants targeted to endogenous human cell sites
a, On-target activities of WT SpCas9, SpCas9-HF1, Cluster 1 and Cluster 2 variants across 24 endogenous human genes, assessed by T7E1 assay; mean and s.e.m. shown; n = at least 3 biologically independent samples (overlaid as white circles). b, WT-normalized endogenous gene disruption data from panel a, for Cluster 1 and 2 variants. Error bars represent median and interquartile ranges of 24 biologically independent samples with the >70% interval of WT activity highlighted in light grey; Cluster 1 (HypaCas9) data from Fig. 3b is replotted for comparison. c–e, Summary of single mismatch tolerance of WT SpCas9, SpCas9-HF1, eSpCas9(1.1), and Cluster 1 and Cluster 2 variants on c, FANCF site 1 d, FANCF sites 4 and 6, and e, FANCF site 2. Percent modification in panels c–e assessed by T7E1 assay; mean and s.e.m. shown for n = at least 3 biologically independent samples (overlaid as white circles).
Extended Data Figure 9
Extended Data Figure 9. Genome-wide specificity profiles of high fidelity SpCas9 variants defined using GUIDE-seq
a, Number of in silico predicted target sites mismatched by ‘n’ positions for six sgRNAs against the reference human genome (hg38) via Cas-OFFinder. b, Assessment of GUIDE-seq dsODN tag integration at the on-target site for each nuclease and guide combination, detected by RFLP assay. c, On-target editing, determined by T7E1 assay; mean and s.e.m.; n = 3 biologically independent samples (overlaid as white circles) for panels b and c. d, dsODN tag-integration efficiency ratios (integration:mutagenesis, from panels b and c) for each nuclease and guide combination, with means and 95% confidence intervals shown for n = 6 biologically independent samples. e, GUIDE-seq genome-wide specificity profiles for WT SpCas9, SpCas9-HF1, eSpCas9(1.1), and HypaCas9 each paired with six different sgRNAs. Mismatched positions in off-target sites are highlighted in color; GUIDE-seq read counts shown to the right of the sequences, which correlate with approximate cleavage efficiency at a given site; blue circles indicate sites with potential alternate alignments due to RNA or DNA bulges (see Supplementary Table 1); yellow circles indicate off-target sites that are only supported by asymmetric GUIDE-seq reads.
Extended Data Figure 10
Extended Data Figure 10. Conformational gating drives targeting accuracy for SpCas9 variants
a–c, Steady state smFRET histograms measuring a, HNH, b, REC2 and c, REC3 conformational states for HypaCas9 bound to on-target and PAM-distal mismatched substrates. Black curves represent a fit to multiple Gaussian peaks. d–e, Steady state smFRET histograms of Cas9 variants bound to PAM distal mismatched substrates were normalized to and subtracted from that of on-target smFRET histograms. This analysis reveals transitions from one FRET population (negative peak, shaded region) to another population (positive peak, unshaded regions) for d, REC3 and e, REC2. f, Measured distances between residues labelled with Cy3/Cy5 FRET dyes for different substrate-bound Cas9 structures. Residue pairs were designed to report conformational changes of the specified domain (HNH, REC2 or REC3). The distances were measured between Cα atoms of the indicated residues for the associated PDB structures.
Figure 1
Figure 1. High-fidelity Cas9 variants enhance cleavage specificity through HNH conformational control
a, Locations of amino acid alterations in existing high-fidelity SpCas9 variants mapped onto the dsDNA-bound SpCas9 crystal structure (PDB ID: 5F9R); HNH domain is omitted for clarity. b, Dissociation constants with mean and s.d. shown; n = 3 independent experiments (overlaid as white circles). c, Cartoon of DNA-immobilized SpCas9 for measuring HNH conformation by smFRET, with DNA target numbering scheme. d–f, smFRET histograms showing HNH conformation with indicated Cas9 variants bound to on-target and mismatched targets using nucleotide numbers diagramed in panel c. Black curves represent a fit to multiple Gaussian peaks.
Figure 2
Figure 2. The alpha-helical lobe regulates HNH domain activation
a, Domain organization of SpCas9ΔREC3. b, On-target DNA cleavage assay using SpCas9ΔREC3 with increasing concentrations of the REC3 domain supplied in trans, resolved by denaturing PAGE; repeated three independent times with similar results. c, Schematic of SpCas9ΔREC3HNH, with REC3 added in trans. Inactive to active structures represent HNH in the sgRNA-bound (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. d, smFRET histograms showing HNH conformation with SpCas9ΔREC3HNH bound to an on-target substrate, with and without REC3. e, Schematic of SpCas9REC2; HNH domain is omitted for clarity. Inactive to active structures represent REC2 in the sgRNA- (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. f–g, smFRET histograms showing REC2 conformation with f, WT SpCas9REC2 and g, SpCas9-HF1REC2 bound to on-target and mismatched targets. For panels d, f and g, black curves represent a fit to multiple Gaussian peaks.
Figure 3
Figure 3. Targeted mutagenesis within the REC3 domain reveals a SpCas9 variant with hyper-accurate behavior in human cells
a, Zoomed image of the REC3 domain and Linker 2 (L2) with amino acids of Cluster variants indicated (PDB ID: 5F9R). Boxed residues indicate amino acids also present in SpCas9-HF1. b, WT-normalized activity of Cas9 variants, using sgRNAs targeting 12 different sites within EGFP. c, WT-normalized endogenous gene disruption activity measured by T7 endonuclease 1 (T7E1) assay across 24 sites. For panels b and c, error bars represent median and interquartile ranges for n = 12 or 24 biologically independent samples, respectively; the interval with > 70% of wild-type activity is highlighted in light grey. d, Activities of WT and high-fidelity Cas9 variants when programmed with singly mismatched sgRNAs against FANCF site 1. e, Activities of Cas9 variants when programmed with singly mismatched sgRNAs against FANCF site 4 and FANCF site 6. f, Histogram of the total number of GUIDE-seq detected off-target sites for Cas9 variants with six different sgRNAs.
Figure 4
Figure 4. Mutating residues involved in proofreading increases the threshold for conformational activation to ensure targeting accuracy
a, DNA cleavage kinetics of SpCas9 variants with the FANCF site 1 on-target and internally mismatched substrates; mean and s.d. shown; n = 3 independent experiments (overlaid as white circles). b, smFRET histograms showing HNH conformation for indicated SpCas9 variants with a FANCF site 1 on-target and mismatched substrate at the 12th position; black curves represent a fit to multiple Gaussian peaks. c, Model for alpha-helical lobe sensing and regulation of the RNA/DNA heteroduplex for HNH activation and cleavage.

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