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Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems

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Post-Transcriptional Gene Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2404))

Abstract

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.

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Acknowledgments

We thank Dr. Chunyu Liao for providing the plasmid expressing the Leptotrichia shahii Cas13a, Ms. Fani Ttofali for providing the plasmid expressing the non-targeting gRNA for LsCas13a, and Ms. Franziska Wimmer for providing feedback on the protocol.

Funding

This work was supported by the DARPA Safe Genes program (HR0011-17-2-0042 to C.L.B.).

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Authors and Affiliations

  1. Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), Würzburg, Germany

    Katharina G. Wandera & Chase L. Beisel

  2. Medical Faculty, University of Würzburg, Würzburg, Germany

    Chase L. Beisel

Authors
  1. Katharina G. Wandera
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  2. Chase L. Beisel
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Corresponding author

Correspondence to Chase L. Beisel .

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Editors and Affiliations

  1. Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy

    Erik Dassi

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Wandera, K.G., Beisel, C.L. (2022). Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 2404. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1851-6_7

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  • DOI: https://doi.org/10.1007/978-1-0716-1851-6_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1850-9

  • Online ISBN: 978-1-0716-1851-6

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