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.