(632b) Next Generation CRISPR-Cas Tools for Post-Transcriptional Gene Regulation in Bacteria

The CRISPR-Cas system provides powerful tools to regulate gene expression in bacteria. Cas9-based systems typically act at the DNA level to activate, repress, or knockout gene targets. However, transcriptional regulation in bacteria has important limitations in its ability to precisely target individual genes for up- or down-regulation. Specifically, CRISPR-Cas9 is unable to regulate single genes in a transcriptional operon. We have shown that targeting of one gene for activation or repression in a multigene operon leads to simultaneous regulation of all genes in the same unit. These limitations greatly restrict our ability to efficiently direct metabolic flux to a desired product for biosynthesis. We have recently engineered Cas13-based RNA targeting tools for use in bacteria. These tools function post-transcriptionally to overcome the limitations of DNA-targeting CRISPR-Cas systems. Here, we characterize new tools leveraging CRISPR-Cas13 that allow for individual targeting of genes in a transcriptional operon for up- and down- regulation to improve microbial biosynthesis.