Rational Engineering of a Modular Bacterial CRISPR-Cas Activation Platform with Expanded Target Range

The ability to control gene expression is a transformative capability that allows to uncover gene function or induce valuable phenotypes and cell fates important for applications in biotechnology and biomedicine. CRISPR-Cas regulators have provided an excellent toolbox for this purpose. Among these, CRISPR activator (CRISPRa) systems turn on transcription through localization of activation domains (AD). However, current bacterial CRISPRa systems still present several challenges. First, different bacterial ADs offer distinct regulatory properties, meaning different AD are often required depending on the target gene or desired application. Second, the bacterial CRISPRa systems need to be targeted to sites located a specific number of nucleotides upstream of the promoter to achieve activation, which critically limits its application in non-synthetic targets. To address these limitations, we adopt a protein engineering approach to create a highly modular and target flexible bacterial CRISPRa system. We identified a novel and strong AD which we harness to explore AD recruitment strategies. Specifically, we evaluated the use of modular protein-protein interaction domains which allows different AD to be encoded on independent and easily exchangeable plasmid elements. Additionally, we evaluated the use of circularly permuted variants of the dCas9 protein to identify variants capable of activating gene expression from distinct positions previously identified as non-activating sites and therefore expand the functional target range. Our modular CRISPRa system provides a more flexible and versatile tool that will expand our ability to activate and control bacterial genomes.