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Structure-based optimization and design of CRISPR protein xCas9 / 生物工程学报
Chinese Journal of Biotechnology ; (12): 1385-1395, 2021.
Article in Chinese | WPRIM | ID: wpr-878640
ABSTRACT
Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Streptococcus pyogenes / Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Cas Systems / Gene Editing / CRISPR-Associated Protein 9 Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2021 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Streptococcus pyogenes / Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Cas Systems / Gene Editing / CRISPR-Associated Protein 9 Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2021 Type: Article