Programming Targeted, Tunable, and Highly Specific Gene Silencing with Native Crispr-Cas Systems

CRISPR-Cas defense systems have proven to be powerful heterologous tools for genome editing and transcriptional control. However, an unexplored avenue is using native CRISPR-Cas systems as convenient tools for the diverse bacteria and archaea that already harbor these systems. Here, we describe the capacity of Type I CRISPR-Cas systems, the most prevalent CRISPR-Cas systems in both bacteria and archaea, for gene regulation. Using the Type I-E CRISPR-Cas system native to Escherichia coli K-12 as a model, we found that this system could be readily converted into a programmable repressor through the deletion of the signature cas3 gene. Plasmid-based expression of arrayed CRISPR RNAs could further enact potent and reversible silencing of multiple endogenous and heterologous genes as well as generate defined cellular phenotypes. Remarkably, we found that increasing the length of the targeting spacer improved gene silencing and enhanced the targeting specificity--in stark contrast to gene silencing with Type II CRISPR-Cas systems and the catalytically dead Cas9. We are currently applying our approach for dynamic control of metabolic pathways and for rapidly elucidating the protospacer-adjacent motif unique to each CRISPR-Cas system. Overall, this general strategy offers a convenient toolkit in synthetic biology for probing and programming gene regulatory networks, particularly in the diversity of undomesticated microorganisms for which few genetic tools exist.