Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements
Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements
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DOI：
10.1038/s41467-020-15454-y
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发表时间：
2020-04-01
影响因子：
16.6
通讯作者：
Zalatan, Jesse G.
中科院分区：
文献类型：
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作者：
Fontana, Jason;Dong, Chen;Zalatan, Jesse G.
在细菌系统中，CRISPR-Cas转录激活（CRISPRa）有可能极大地扩展我们调节基因表达的能力，但我们缺乏设计有效gRNA靶点的预测规则。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狭窄的2-4个碱基窗口，其周期性对应于DNA的一个螺旋旋转，在TSS上游跨越约40个碱基，中心约为80个碱基。然而，我们也发现了两个特征，表明其具有广泛的潜力：CRISPRa在广泛的sigma（70）家族启动子中有效，并且扩展的PAM dCas9允许激活不能被化脓性链球菌dCas9激活的启动子。这些结果为未来进一步扩大和推广细菌CRISPRa范围的工程努力提供了路线图。
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 similar to 40 bases and centered similar to 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 sigma(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.