(556a) Exploring Streptococci Cas9 Orthologs with Distinct PAM-Binding Motifs to Develop a Titratable Bacterial Gene Regulator

The CRISPR-Cas9s have been developed as versatile tools for programmable genome editing and gene expression control. However, their targetable sequences and targeting efficacies are restricted by the strict requirement on a specific protospacer adjacent motif (PAM) sequence. The SpCas9 from S. pyogenes, representing most Streptococci Cas9s, requires a NGG PAM by its signature RxR PAM-binding motif. In this study, we reveal that three rarely reported PAM-binding motifs among Streptococci Cas9 orthologs generate distinguished PAM specificities. Rational site-directed mutagenesis of the SeCas9 from S. equinus with a different motif naturally preferring NAG PAM produces a variant with expanded PAM range. With this, we further develop a PAM-dependent titratable gene regulator by fusing an ω factor with the deactivated SedCas9 variant. Applying such a regulator for the metabolic control of endogenous genes in Escherichia coli leads to higher 4-hydroxycoumarin production. These results demonstrate the PAM diversity of closely-related Streptococci Cas9 orthologs and enrich the PAM-flexible Cas9 toolkits for broader applications.