Combined Computational–Experimental Approach to Explore the Molecular Mechanism of SaCas9 with a Broadened DNA Targeting Range

• Published in: Journal of the American Chemical Society Vol. 141; no. 16; pp. 6545 - 6552
• Main Authors: Luan, Binquan, Xu, Guangxue, Feng, Mei, Cong, Le, Zhou, Ruhong
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.04.2019
• Subjects: CRISPR-Associated Protein 9 - chemistry; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; DNA - genetics; Molecular Dynamics Simulation; Mutation; Protein Conformation; Protein Engineering; Staphylococcus aureus - enzymology
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.8b13144

Despite accelerating development of CRISPR technology, there remains high demand for further interrogation of its fundamental biology. This is particularly fascinating as new improved CRISPR tools were artificially engineered to harbor beneficial features but often lack mechanistic explanation. SaCas9, a minimal Cas9 ideal for in vivo applications, suffers from long protospacer adjacent motif (PAM), which prompted effort on mutant KKH SaCas9 with relaxed PAM requirement. Leveraging structure-based molecular dynamics simulation, free-energy perturbation, and targeted experimentation, we developed a workflow for probing SaCas9 and a series of its variants, revealing intriguing dynamics of PAM recognition and the molecular mechanism of KKH mutations. Furthermore, we deployed this approach to design and validate new mutant SaCas9, SaCas9-NR and SaCas9-RL, with enhanced targeting range distinctive from the KKH mutant and improved activity in mammalian cells. Overall, our approach provides a dynamic understanding of SaCas9 PAM recognition and a new gateway to enlighten rationally designed Cas9 variants harboring novel properties.