Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

TLDR

Artificial control of transcription is essential for studying gene function and building synthetic gene networks, and Cas9 is an RNA‑guided DNA nuclease used in CRISPR‑Cas immunity. The authors aim to develop a Cas9 mutant that can be programmed to repress or terminate transcription. They engineered a DNA‑binding Cas9 mutant and fused it with the RNAP omega subunit to enable programmable repression, termination, and activation of transcription. The technology allows simple, efficient modulation of gene expression, benefiting gene‑network studies and synthetic biology applications.

Abstract

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.