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 σ⁷⁰-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.

在细菌系统中，CRISPR-Cas 转录激活 (CRISPRa) 有潜力显着扩展我们调节基因表达的能力，但我们缺乏设计有效 gRNA 靶位点的预测规则。在这里，我们确定了细菌启动子的多个特征，这些特征对 CRISPRa 靶位点提出了严格的要求。值得注意的是，我们观察到有效位点的狭窄 2-4 个碱基窗口，其周期性对应于 DNA 的一圈螺旋，跨越约 40 个碱基，并以 TSS 上游的约 80 个碱基为中心。然而，我们还发现了两个特征，表明其具有广泛的潜力：CRISPRa 对广泛的 σ ⁷⁰家族启动子有效，并且扩展的 PAM dCas9 允许激活化脓性链球菌dCas9 无法激活的启动子。这些结果为未来进一步扩展和推广细菌 CRISPRa 范围的工程工作提供了路线图。