(500c) Repurposing Endogenous Crispr-Cas Bacterial Immune Systems for Programmable Gene Repression

CRISPR-Cas systems offer powerful and versatile biomolecular tools with many applications including genome editing, gene regulation, sequence-specific antimicrobials, and imaging. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness endogenous CRISPR-Cas systems as convenient tools in these organisms. We report that the Type I-E CRISPR-Cas system in the bacterium Escherichia coli can be co-opted for programmable transcriptional repression. Deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible and multiplexable silencing of heterologous and endogenous genes.

Utilizing this platform, we explored how the length of the CRISPR RNA impacts the form and function of the Type I-E effector protein complex called Cascade. We discovered that the length of the Type I-E CRISPR RNA molecule is not fixed and can be extended, altering the stoichiometry of the Cascade effector complex while preserving functionality. Interestingly, altering RNA length can elicit significant improvement in transcriptional silencing efficiency, but only for particular target locations. These findings heighten our understanding of CRISPR-Cas systems as they continue to increase in popularity and may offer insight into designing tunable transcriptional regulators.