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CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes

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Phytochromes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2026))

Abstract

Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.

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Change history

  • 22 June 2021

    In the original version of this book, Chapter 9 was inadvertently published with few errors in Table 2 data. This has now been rectified in this revised version of the book.

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Acknowledgments

This work was supported by DFG grant Hu702/5 to JH and by French ANR grant ANR11-BTBR-0001-GENIUS to NF. The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). We thank Pierre François Perroud for his expertise and advice in the context of moss transformation.

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Authors and Affiliations

  1. Institute for Plant Physiology, Justus Liebig University, Giessen, Germany

    Anna Lena Ermert, Fabian Stahl, Tanja Gans & Jon Hughes

  2. Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France

    Fabien Nogué

Authors
  1. Anna Lena Ermert
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  2. Fabien Nogué
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  3. Fabian Stahl
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  4. Tanja Gans
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  5. Jon Hughes
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Corresponding authors

Correspondence to Fabien Nogué or Jon Hughes .

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Editors and Affiliations

  1. Faculty of Biology, Institute of Biology II and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

    Andreas Hiltbrunner

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Ermert, A.L., Nogué, F., Stahl, F., Gans, T., Hughes, J. (2019). CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes. In: Hiltbrunner, A. (eds) Phytochromes. Methods in Molecular Biology, vol 2026. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9612-4_20

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  • DOI: https://doi.org/10.1007/978-1-4939-9612-4_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9611-7

  • Online ISBN: 978-1-4939-9612-4

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