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

Jason Fontana, Chen Dong, Cholpisit Kiattisewee, Venkata P. Chavali, Benjamin I. Tickman, James M. Carothers () and Jesse G. Zalatan ()
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Jason Fontana: University of Washington
Chen Dong: University of Washington
Cholpisit Kiattisewee: University of Washington
Venkata P. Chavali: University of Washington
Benjamin I. Tickman: University of Washington
James M. Carothers: University of Washington
Jesse G. Zalatan: University of Washington

Nature Communications, 2020, vol. 11, issue 1, 1-11

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 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.

Date: 2020
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DOI: 10.1038/s41467-020-15454-y

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