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RNA Nanostructures pp 341–357Cite as

CRISPR-Cas RNA Scaffolds for Transcriptional Programming in Yeast

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1632))

Abstract

CRISPR scaffold RNAs (scRNAs) provide a modular system for locus-specific transcriptional programming. scRNAs are generated by extending CRISPR guide RNA sequences with domains that recruit RNA-binding proteins, thus physically linking DNA binding and protein recruitment activities. A single scRNA molecule encodes information about the target locus and instructions about what regulatory function to execute at that locus. Sets of scRNA constructs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. Such programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point, and this approach has been recently applied to flexibly redirect flux through a complex branched metabolic pathway in yeast. This protocol describes how to construct multi-scRNA transcriptional programs in yeast, including target site selection, cloning strategies, and yeast engineering.

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Acknowledgments

The author would like Brian Fan, Robin Kirkpatrick, and Jingwen Sun for validating and reproducing several of the methods described here, as well as members of the Lim, Qi, Dueber, and Weissman labs for technical support and discussions. This work was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

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Authors and Affiliations

  1. Department of Chemistry, University of Washington, 36 Bagley Hall, Seattle, WA, 98195, USA

    Jesse G. Zalatan

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  1. Jesse G. Zalatan
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Correspondence to Jesse G. Zalatan .

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Editors and Affiliations

  1. Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Frederick, Maryland, USA

    Eckart Bindewald

  2. RNA Structure and Design Section, RNA Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA

    Bruce A. Shapiro

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Zalatan, J.G. (2017). CRISPR-Cas RNA Scaffolds for Transcriptional Programming in Yeast. In: Bindewald, E., Shapiro, B. (eds) RNA Nanostructures . Methods in Molecular Biology, vol 1632. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7138-1_22

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  • DOI: https://doi.org/10.1007/978-1-4939-7138-1_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7137-4

  • Online ISBN: 978-1-4939-7138-1

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