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

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. SaCa...

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Published inJournal 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
LanguageEnglish
Published United States American Chemical Society 24.04.2019
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Abstract 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.
AbstractList 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.
Author Xu, Guangxue
Feng, Mei
Cong, Le
Luan, Binquan
Zhou, Ruhong
AuthorAffiliation Computational Biological Center
Institute of Quantitative Biology, Department of Physics
Tsinghua University
Department of Pathology, Department of Genetics
School of Life Sciences
Zhejiang University
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Snippet Despite accelerating development of CRISPR technology, there remains high demand for further interrogation of its fundamental biology. This is particularly...
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SubjectTerms 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
Title Combined Computational–Experimental Approach to Explore the Molecular Mechanism of SaCas9 with a Broadened DNA Targeting Range
URI http://dx.doi.org/10.1021/jacs.8b13144
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Volume 141
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