731. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects

By B. Kleinstiver, V. Pattanayak, Michelle S. Prew, Shengdar Q. Tsai, Nhu T. Nguyen, J. Keith Joung. 2016

845 citations recorded by the source corpus.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations at genomic locations that resemble the intended target. These so-called off-target effects can confound research applications and are important considerations for potential therapeutic use. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) have thus far proven to be imperfect by possessing only partial efficacy and/or other limitations that constrain their use. Here we describe a high-fidelity variant of SpCas9, called SpCas9-HF1, that contains alterations in the amino acid sequence designed to reduce non-specific contacts to the target strand DNA. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of the 37 single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with eight different sgRNAs targeted to standard non-repetitive sequences in human cells, SpCas9-HF1 rendered all or nearly all off-target events imperceptible 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 important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Our findings also suggest a general strategy for improving or optimizing the genome-wide specificities of other Cas9 orthologues and engineered variants.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a2ed64f67eccc49ff375c7abace4379a6df4b180

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In Molecular Biology

Citations in the corpus

Crystal structure of Cpf1 in complex with guide RNA and target DNA (2016, background): “Structure-guided engineering and direct evolution approaches have led to variants of Cas9 with enhanced target specificity (Slaymaker et al., 2016; Kleinstiver et al., 2016) or altered PAM requirements (Kleinstiver et al., 2015a, 2015b).”
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems (2016, methodology): “Finally, structure-guided engineering has been used to mutate specific residues in Cas9, to weaken the interaction with the nontarget strand or to decrease nonspecific interactions with the target DNA site, favoring cleavage at sites that are perfectly complementary to the guide RNA and reducing off”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “Cas9 mutants have also been designed to reduce non-specific DNA contacts by weakening binding of 1240 C. A. LINO ET AL. the target DNA strand (Kleinstiver et al., 2016) or the non-target DNA strand (Slaymaker et al., 2016) to Cas9 while maintaining robust on-target cleavage.”
Cas-analyzer: an online tool for assessing genome editing results using NGS data (2016, background): “Contact: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online.”