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Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA

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CRISPR Guide RNA Design

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

Abstract

CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. It is also now appreciated that chemically modified nucleic acids can substitute for unmodified nucleotides in guide RNAs, providing protection from exonuclease degradation and improving gene editing efficiency. CRISPR-Cpf1 unlike CRISPR-Cas9, has a substantially lower propensity for off-target genomic cleavage, making it a preferred gene editing system for many applications. Here we provide a detailed protocol for use of CRISPR-Cpf1 and chemically modified guide RNAs in cell lines, outlining the steps from designing guide RNAs to a target gene of interest, delivery and expression in cells, and analysis of gene editing events.

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Author information

Authors and Affiliations

  1. Ionis Pharmaceuticals Inc., Carlsbad, CA, USA

    Moira A. McMahon & Meghdad Rahdar

Authors
  1. Moira A. McMahon
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  2. Meghdad Rahdar
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Corresponding author

Correspondence to Meghdad Rahdar .

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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McMahon, M.A., Rahdar, M. (2021). Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA. 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_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0686-5

  • Online ISBN: 978-1-0716-0687-2

  • eBook Packages: Springer Protocols

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