2016
DOI: 10.1038/nature16526
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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-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 SpC… Show more

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Cited by 2,251 publications
(2,012 citation statements)
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“…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.…”
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confidence: 98%
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“…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.…”
mentioning
confidence: 98%
“…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 59 . 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.…”
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confidence: 99%
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