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 gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems. Overall design: K562 cells transduced with an Sp-Cas9 nuclease variant and sgRNA library targeting CD45 were pooled together prior to antibody staining and sorting based on the expression of the target gene. A475 cells transduced with an Sp-dCas9-VPR transcriptional activator variant and sgRNA library targeting CD45 were pooled together prior to antibody staining and sorting based on the expression of the target gene. sgRNA distribution in the cell pool prior to sorting was used as a reference.