Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity.

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 subst

SUBMITTER: Hand TH 

PROVIDER: S-EPMC6525624 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature