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In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems

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CRISPR Guide RNA Design

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

Abstract

CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.

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Acknowledgments

This work was supported by NSERC Discovery/Discovery Accelerator Supplement (RGPIN-2016-0638) and CIHR (PS-408552) grants to B.P.H. We thank Dr. Juan Jovel, Sudip Subedi, and the members of the Applied Genomics Core at the University of Alberta for their technical assistance with next-generation sequencing. We thank Bio-Synthesis Inc. (Texas) for providing the 2′OMePACE-modified guide RNA.

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Authors and Affiliations

  1. Department of Pharmacology, University of Alberta, Edmonton, AB, Canada

    Christopher R. Cromwell & Basil P. Hubbard

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  1. Christopher R. Cromwell
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  2. Basil P. Hubbard
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Corresponding author

Correspondence to Basil P. Hubbard .

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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Cromwell, C.R., Hubbard, B.P. (2021). In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems. 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_12

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  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_12

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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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