High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

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High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

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Title: High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets
Author: Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu; Zheng, Zongli; Joung, J. Keith

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Citation: Kleinstiver, 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.
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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.
Published Version: doi:10.1038/nature16526
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851738/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:27822297
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