A High-Throughput Growth-Based Screen for CRISPR Pams Reveals Novel SpyCas9 Targeting Rules in Bacteria

We developed and characterized a novel growth-based positive PAM assay to characterize CRISPR-Cas systems. This PAM screen manipulated the E. coli carbon metabolism so growth was only observed in the presence of D-xylose if a functional PAM was recognized on a replicated plasmid. The assay was characterized with the ubiquitous Streptococcus pyogenes Cas9 nuclease and instead of presenting its most functional PAMs, the screen enriched for weakly recognized NNGG PAMs. We demonstrated that S. pyogenes Cas9 is able to functionally target an NNGG motif using both transcriptional repression and plasmid clearance in a bacterial host. This PAM proved to be less functional than the canonical NGG motif, but still showed significant activity over non-PAMs. Altogether, we develped a new tool to discover the least function PAMs for CRISPR-Cas systems in bacteria and expanded the targeting rules for S. pyogenes Cas9.