Mechanistic insight on a chimeric Cas9 protein's specificity for DNA target with 5 '-NAA-3' PAM

Abstract

Numerous protein variants have been made to expand the repertoire of CRISPR-Cas nucleases that can recognize protospacer-adjacent motifs (PAMs) other than the canonical NGG discovered in wild-type Streptococcus pyogenes. While Cas nuclease engineering has largely yielded proteins with enhanced specificity for NGG and variations on G-containing PAMs, we were able to construct a chimeric Cas protein with consistent specificity for a 5'-NAA-3' PAM by rationally combining the PAM-interacting domain of Streptococcus macacae with the S. pyogenes Cas9 scaffold. We have been able to demonstrate during in vitro incubations that our chimeric protein is capable of cleaving dsDNA with an NAA PAM, but a deeper biochemical understanding of the nature of these new chimeric proteins' binding and cleavage activities is of paramount importance for their practical use. Here, we use of the principles of enzyme kinetics to investigate our chimeric protein's comparative efficiency to Cas12a and the biophysical mechanism by which our grafted S. macacae segment works synergistically with the S. pyogenes Cas9 scaffold to cleave target DNA with an NAA PAM. We show that SpySmacCas9 does not bind or cleave at rates comparable to Casl2a, but its overall performance rivals that of wild-type SpyCas9 with a new PAM preference.