期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 116, 期 42, 页码 20969-20976出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1906843116
关键词
CRISPR-Cas9; SaCas9; off-target
资金
- Ming Wai Lau Centre of Reparative Medicine of Karolinska Institutet
- City University of Hong Kong
- National Natural Science Foundation of China [81672098, 81770099]
- Swedish Research Council [2016-02830]
- Innovation and Technology Fund of Hong Kong Government [9440153]
- Hong Kong Health and Medical Research Fund [05160296]
- Hong Kong Research Grants Council [21101218]
- Shenzhen Science and Technology Innovation Fund [JCYJ20170413115637100, JCYJ20170412152916724]
- Sanming Project of Medicine in Shenzhen [SZSM201811092]
- Swedish Research Council [2016-02830] Funding Source: Swedish Research Council
- Vinnova [2016-02830] Funding Source: Vinnova
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 ontarget efficiency. One engineered variant, referred to as SaCas9HF, 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 ontarget 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据