Synthetic CRISPR Tools for Information Processing in Bacteria

Abstract

CRISPR Cas tools enable programmable control over gene expression and editing in bacteria. However, CRISPR activation in bacteria faces challenges including strict target site requirements, unreliable performance across native genes, and interference from native gene regulation. This work aims to overcome these challenges by creating versatile and robust CRISPR tools in bacteria. We systematically evaluated and developed PAM-flexible dCas9 variants, engineered bacterial activators, and mRNA-responsive molecular switches. First, with PAM-flexible dCas9 variants, we observe activation at previously inaccessible gene targets, and we observe a tradeoff between fold-activation and PAM flexibility. Our work provides a framework to choose the most effective dCas9 variant for a given endogenous gene target. Next, we characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary upstream of the gene start site and combinations of activators often resulted in antagonistic behavior. Last, we combined CRISPR tools to create mRNA-responsive gene switches for molecular recording and programmable, permanent expression changes. This dual-Cas9 approach uses reprogrammable RNAs to sense mRNA signals and convert them into gene activation, achieving high editing efficiency and high activation of a fluorescent reporter. These advancements enhance CRISPR tools for dynamic, programmable regulation and highlight future strategies for next-generation bacterial gene circuits.