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Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System

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Part of the Methods in Molecular Biology book series (MIMB,volume 2518)

Abstract

CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.

Key words

  • CRISPR activation
  • CRISPR-Cas regulators
  • Transcription activation

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  • DOI: 10.1007/978-1-0716-2421-0_12
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Acknowledgments

Thanks goes to Welch Foundation [C-1982–20190330 to J.C.] and Alfred P. Sloan Research Fellowship [FG-2018-10500 to J.C.]; J.C. is an Alfred P. Sloan Research Fellow.

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Correspondence to James Chappell .

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Villegas Kcam, M.C., Chappell, J. (2022). Design, Characterization, and Application of Targeted Gene Activation in Bacteria Using a Modular CRISPRa System. In: Chappell, J., Takahashi, M.K. (eds) Riboregulator Design and Analysis. Methods in Molecular Biology, vol 2518. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2421-0_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2421-0_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2420-3

  • Online ISBN: 978-1-0716-2421-0

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