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

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journal

5 Scopus Citations
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Detail(s)

Original languageEnglish
Pages (from-to)20969-20976
Journal / PublicationProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number42
Online published30 Sep 2019
Publication statusPublished - 15 Oct 2019

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.

Research Area(s)

  • CRISPR-Cas9, Off-target, SaCas9

Citation Format(s)

Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. / Tan, Yuanyan; Chu, Athena H.Y.; Bao, Siyu; Hoang, Duc Anh; Kebede, Firaol Tamiru; Xiong, Wenjun; Ji, Mingfang; Shi, Jiahai; Zheng, Zongli.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 42, 15.10.2019, p. 20969-20976.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journal