(353c) Repurposing Crispr-Cas Systems for “Built-in” Transcriptional Repression

CRISPR-Cas systems—RNA-directed immune 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 “built-in” tools for the diverse bacteria and archaea harboring these systems. While biotechnological interest has overwhelming focused on Type II CRISPR-Cas systems and its Cas9 endonuclease, these systems are the least common and require targeted point mutations to cas9 to achieve transcriptional control. Therefore, we explored the capacity of endogenous Type I CRISPR-Cas systems, the most common type of CRISPR-Cas systems, to achieve transcriptional regulation.

Using the Type I-E CRISPR-Cas system in the bacterium Escherichia coli as a model, we found that these systems could be readily converted into programmable repressors through the one-step deletion of the signature cas3 gene. Furthermore, plasmid-based expression of CRISPR RNAs revealed potent silencing of multiple endogenous and heterologous genes, design rules for potent and multiplexed gene targeting, and the generation of defined cellular phenotypes. Overall, this general strategy could provide indispensible “built-in” tools for diverse non-model microorganisms and offers a framework for the discovery of Type I CRISPR-Cas systems that naturally regulate gene expression.