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-targets of the broadly 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 harboring 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. Strikingly, 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-targets induced by SpCas9-HF1 were not detected. 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 RNA-guided nucleases.

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…However, when SpCas9-HF1 HNH was bound to a substrate with a single mismatch at the PAM-distal end (20-20 bp mm), stable docking of the HNH nuclease was entirely ablated (Figure 1e). In addition, eSpCas9(1.1) HNH and other high fidelity variants 8,9 reduced the HNH active state in the presence of mismatches (Figure 1f, Extended Data Figure 2c–d). We therefore propose that high fidelity variants of Cas9 reduce off-target cleavage by raising the threshold for HNH conformational activation when bound to DNA substrates.…”

“…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 were unknown 5–9 . Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state 10 when bound to mismatched targets.…”

“…Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of SpCas9-HF1 and eSpCas9(1.1) variants that contain amino acid substitutions predicted to weaken the energetics of target site recognition and cleavage 8,9 (Figure 1a). Biochemically, we found that these Cas9 variants cleaved the on-target DNA with rates similar to that of wild-type (WT) SpCas9, whereas their cleavage activity was significantly reduced on substrates bearing mismatches (Extended Data Figures 1a, 2a).…”

“…Biochemically, we found that these Cas9 variants cleaved the on-target DNA with rates similar to that of wild-type (WT) SpCas9, whereas their cleavage activity was significantly reduced on substrates bearing mismatches (Extended Data Figures 1a, 2a). To test the hypothesis that SpCas9 with its single-guide RNA (sgRNA) might exhibit a greater affinity for its target than is required for effective recognition 9,11 , we measured DNA binding affinity and cleavage of SpCas9-HF1 and eSpCas9(1.1) variants. Contrary to a potential hypothesis that mutating these charged residues to alanine weakens target binding 11 , the affinities of these variants for on-target and PAM-distal mismatched substrates were similar to WT SpCas9 (Figure 1b, Extended Data Figures 1a, 2b), indicating that cleavage specificity is improved through a mechanism distinct from a reduction of target binding affinity 11 .…”

“…We identified five clusters of residues containing conserved amino acids within 5 Å of the RNA/DNA interface, four of which are located within REC3 and one in the HNH-RuvC Linker 2 (L2) (Figure 3a, Extended Data Figure 5). Alone or in combination with Q926A, a substitution within L2 that confers higher specificity 9 , we generated alanine substitutions for each residue within the five different clusters of amino acids (Clusters 1–5 ± Q926A) (Figure 3a). We tested whether these cluster mutations affect cleavage accuracy and equilibrium binding in vitro , and found that Cluster 1 alone and Cluster 2 + Q926A suppressed off-target cleavage while retaining target binding affinities comparable to WT (Extended Data Figure 6).…”