(632a) Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants

CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective modulation. We demonstrate that expanded PAM dCas9 variants can increase the number of targetable CRISPRa sites in bacteria and systematically evaluate the capabilities of different dCas9 variants. In particular, we observed that dxCas9-NG provided a high dynamic range of gene activation for NGN PAMs while dSpRY permitted modest activity across almost any PAM sequence. However, this improved PAM-flexibility comes with the tradeoff that CRISPR interference (CRISPRi) becomes less effective. Weaker CRISPRi gene repression can be partially rescued by targeting multiple sites in the gene of interest. By systematically characterizing the properties of expanded PAM dCas9 variants in bacterial CRISPRa/i, we provide a framework to choose the most effective variant for a given gene target. This toolbox of dCas9 variants will further expand the utility of CRISPRa/i in bacterial systems for a broad range of applications including genome-wide screens and metabolic engineering.