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

BP Kleinstiver, V Pattanayak, MS Prew, SQ Tsai… - Nature, 2016 - nature.com
Nature, 2016nature.com
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> …
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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