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 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.

Directed evolution studies of a thermophilic Type II-C Cas9

“…6). Site-directed mutation of Glu840 to a smaller amino acid, alanine, indeed had a substantially reduced survival rate (Hand et al, in review) (Fig. 6).…”

“…We showed previously that PAM-proximal mismatches between the protospacer and the guide RNA severely impaired AceCas9 cleavage activity, leading to death of the bacteria in the presence of arabinose (Hand et al, in review; Tsui et al, 2017). The lack of survival with PAM-proximal mismatches prompted us to search for mutant AceCas9s that could rescue this phenotype.…”

“…These variants, but not the WT AceCas9, yielded high cell survival rates in the presence ofa −1 position mismatch. This led us to hypothesize the role of charge and size in influencing AceCas9 sensitivity to mismatches at PAM-proximal locations (Hand et al, in review) (Fig. 6).…”

Catalytically Enhanced Cas9 through Directed Protein Evolution

“…Mutations that disrupt one of the two hinge helices lowered the DNA cleavage activity of SpyCas9 27 .These detailed mechanistic studies suggest that the allosteric hinge is also a potential target for engineering CECas9 33 . However, protein engineering by rational design to select for CECas9 variants would be laborious and have limited chances of successes.We previously described a toxicity-based bacterial survival assay that is amenable for a library-based directed protein evolution selection 34,35 . In this approach, a library of plasmids encoding a collection of Cas9 variants is transformed into bacterial cells harboring a plasmid expressing the toxic protein ccdB under the induction by arabinose.…”

“…In bacterial cells, while AceCas9 targeting a 24-nt protospacer causes near one hundred percent survival in the ccdB-based cell survival assay, that targeting a 20-nt protospacer has less than 0.2% rate of survival ( Figure S1). In a typical transformation experiment, therefore, no surviving colonies would be observed when the wild-type AceCas9 is introduced to electro-competent E. coli cells harboring the ccdB-plasmid with a 20-nt protospacer 35,36 . We took advantage of this selection feature in identifying AceCECas9.We hypothesized that the allosteric hinge can be engineered to allow a faster HNH conformational sampling, which leads to faster RuvC cleavage, resulting in more efficient formation of double-stranded breaks.…”