Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

TLDR

CRISPR‑Cas transcriptional activation (CRISPRa) promises to greatly expand bacterial gene regulation, yet no predictive rules exist for designing effective gRNA target sites. The study aims to identify promoter features that impose stringent requirements on CRISPRa target sites. The authors analyze bacterial promoter characteristics to determine these constraints. They find that effective CRISPRa sites are confined to narrow 2–4 base windows with a one‑helical‑turn periodicity, spanning ~40 bases centered ~80 bases upstream of the TSS, yet CRISPRa works across many σ70 promoters and with an expanded PAM dCas9, offering a roadmap for broader bacterial CRISPRa.

Abstract

Abstract In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2–4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ 70 -family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.