High-fidelity SaCas9 identified by directional screening in human cells

• Published in: PLoS biology 2020-07, Vol.18 (7), p.e3000747-e3000747
• Main Authors: Xie, Haihua, Ge, Xianglian, Yang, Fayu, Wang, Bang, Li, Shuang, Duan, Jinzhi, Lv, Xiujuan, Cheng, Congsheng, Song, Zongming, Liu, Changbao, Zhao, Junzhao, Zhang, Yu, Wu, Jinyu, Gao, Caixia, Zhang, Jinwei, Gu, Feng
• Format: Article
• Language: English
• Subjects: Accuracy; Bacterial Proteins - metabolism; Biology and Life Sciences; Cells; CRISPR; CRISPR-Associated Protein 9 - metabolism; Deoxyribonucleic acid; Diabetes; DNA; Editing; Engineering and Technology; Fidelity; Funding; Gene Library; Genetic aspects; Genetic Engineering; Genetic Loci; Genetic variation; Genome editing; Genome, Human; Genomes; Genomics; HEK293 Cells; Hospitals; Humans; Identification and classification; Kidney diseases; Laboratories; Methods; Methods and Resources; Molecular biology; Mutation; Nucleotides; Nucleotides - genetics; Optometry; Phenotype; Reproducibility of Results; Research and analysis methods; Ribonucleic acid; RNA; Screening; Software; Staphylococcus aureus - metabolism; Toolkits; Transcriptional Activation - genetics
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.3000747

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.