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Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity.


ABSTRACT: RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

SUBMITTER: Tan Y 

PROVIDER: S-EPMC6800346 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Rationally engineered <i>Staphylococcus aureus</i> Cas9 nucleases with high genome-wide specificity.

Tan Yuanyan Y   Chu Athena H Y AHY   Bao Siyu S   Hoang Duc Anh DA   Kebede Firaol Tamiru FT   Xiong Wenjun W   Ji Mingfang M   Shi Jiahai J   Zheng Zongli Z  

Proceedings of the National Academy of Sciences of the United States of America 20190930 42


RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from <i>Staphylococcus aureus</i> (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, ref  ...[more]

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