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Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA–ncRNA Expression Constructs

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CRISPR Guide RNA Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2162))

Abstract

CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA–ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA “cargo” domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA–ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA–ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of “best practices” for the design and implementation of CRISPR-Display experiments.

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Acknowledgments

I thank Yasemin Sancak for her tireless assistance in the preparation of the manuscript, David Hendrickson and Danielle Tenen for sharing their technical expertise in fRIP, John Rinn, Ezgi Hacisuleyman, and Scott Younger for their assistance in developing CRISPR-Display, and Philipp Maass for his assistance with CLING.

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Authors and Affiliations

  1. Department of Pharmacology, University of Washington, Seattle, WA, USA

    David M. Shechner

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  1. David M. Shechner
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Correspondence to David M. Shechner .

Editor information

Editors and Affiliations

  1. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Tudor A. Fulga

  2. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    David J. H. F. Knapp

  3. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Quentin R. V. Ferry

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Shechner, D.M. (2021). Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA–ncRNA Expression Constructs. In: Fulga, T.A., Knapp, D.J.H.F., Ferry, Q.R.V. (eds) CRISPR Guide RNA Design. Methods in Molecular Biology, vol 2162. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0687-2_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0686-5

  • Online ISBN: 978-1-0716-0687-2

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