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A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes

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Book cover Cell-Free Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2433))

Abstract

Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.

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Acknowledgments

This work was supported by the SPP 2141 priority program of the Deutsche Forschungsgemeinschaft (BE 6703/1-1 to C.L.B.). A portion of this research was performed under the JGI-EMSL Collaborative Science Initiative and used resources at the DOE Joint Genome Institute and the Environmental Molecular Science Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL).

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Authors and Affiliations

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

    Franziska Wimmer, Frank Englert & Chase L. Beisel

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

    Chase L. Beisel

Authors
  1. Franziska Wimmer
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  2. Frank Englert
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  3. Chase L. Beisel
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Corresponding author

Correspondence to Chase L. Beisel .

Editor information

Editors and Affiliations

  1. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, and Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Ashty S. Karim

  2. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Center for Synthetic Biology, Robert H. Lurie Comprehensive Cancer Center, and Simpson Querrey Institute, Northwestern University, Evanston, IL, USA

    Michael C. Jewett

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Wimmer, F., Englert, F., Beisel, C.L. (2022). A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_24

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  • DOI: https://doi.org/10.1007/978-1-0716-1998-8_24

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1997-1

  • Online ISBN: 978-1-0716-1998-8

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