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

Author

Listed:
  • 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
    University of Washington
    University of Washington)

  • Jesse G. Zalatan

    (University of Washington
    University of Washington
    University of Washington)

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.

Suggested Citation

  • Jason Fontana & Chen Dong & Cholpisit Kiattisewee & Venkata P. Chavali & Benjamin I. Tickman & James M. Carothers & Jesse G. Zalatan, 2020. "Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-15454-y
    DOI: 10.1038/s41467-020-15454-y
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    Cited by:

    1. Jason Fontana & David Sparkman-Yager & Ian Faulkner & Ryan Cardiff & Cholpisit Kiattisewee & Aria Walls & Tommy G. Primo & Patrick C. Kinnunen & Hector Garcia Martin & Jesse G. Zalatan & James M. Caro, 2024. "Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    2. Rory L. Williams & Richard M. Murray, 2022. "Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli," Nature Communications, Nature, vol. 13(1), pages 1-15, December.

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