ArticleACS synthetic biology2018
Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity.
Article in ACS synthetic biology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
4 citing papers in PubMed.
- Coupled catalytic states and the role of metal coordination in Cas9.Nature catalysis · 2023Article
- Catalytically Enhanced Cas9 through Directed Protein Evolution.The CRISPR journal · 2021Article
- The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.Nature communications · 2020Article
- Directed evolution studies of a thermophilic Type II-C Cas9.Methods in enzymology · 2019Article
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Authors and funding
6 authors.
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Abstract
Despite being utilized widely in genome sciences, CRISPR-Cas9 remains limited in achieving high fidelity in cleaving DNA. A better understanding of the molecular basis of Cas9 holds the key to improve Cas9-based tools. We employed direct evolution and in vitro characterizations to explore structural parameters that impact the specificity of the thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). By identifying variants that are able to cleave mismatched protospacers within the seed region, we found a critical role of the phosphate lock residues in substrate specificity in a manner that depends on their sizes and charges. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to the guide-DNA mismatches. An increase in size of the substituted residues further reduces the sensitivity to mismatches at the first position of the protospacer. Our findings identify the phosphate lock residues as an important site for tuning the specificity and catalytic efficiency of Cas9.
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