Programmable CRISPR-Cas transcriptional activation in bacteria.

Area of Science:

• Synthetic Biology
• Microbiology
• Molecular Biology

Background:

• CRISPR-Cas systems offer programmable gene regulation.
• CRISPR-mediated gene activation (CRISPRa) is well-established in eukaryotes but challenging in bacteria.

Purpose of the Study:

• To develop a generalizable platform for screening and selecting functional bacterial CRISPR-Cas transcription activators.
• To engineer a novel bacterial CRISPRa system for enhanced gene activation and repression.

Main Methods:

• Developed a platform for screening bacterial CRISPR-Cas transcription activators.
• Employed directed evolution to optimize a novel activator, dCas9-AsiA.
• Tested dCas9-AsiA for gene activation and repression in E. coli and other bacteria.

Main Results:

• Identified dCas9-AsiA, a novel CRISPR activator enhancing gene expression over 200-fold.
• Demonstrated simultaneous activation and repression of bacterial regulons using evolved dCas9-AsiA.
• Discovered hundreds of inducible promoters for dCas9-AsiA, creating a resource for genetic engineering.
• Successfully ported dCas9-AsiA to other clinically and industrially relevant bacteria.

Conclusions:

• The developed platform and dCas9-AsiA expand the toolbox for programmable gene regulation in bacteria.
• This work facilitates the engineering of bacterial CRISPR-based gene regulators for diverse applications.