High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets
Joung, J. Keith
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CitationKleinstiver, Benjamin P., Vikram Pattanayak, Michelle S. Prew, Shengdar Q. Tsai, Nhu Nguyen, Zongli Zheng, and J. Keith Joung. 2015. “High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets.” Nature 529 (7587): 490-495. doi:10.1038/nature16526. http://dx.doi.org/10.1038/nature16526.
AbstractCRISPR-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.
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