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CRISPR pp 233–250Cite as

Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1311))

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.

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Acknowledgements

This research was supported by the Israel Science Foundation grant 611/10 to U.Q., and the Marie Curie International Reintegration Grant PIRG-2010-266717 to R.E.

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Authors and Affiliations

  1. Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel

    Ido Yosef, Moran G. Goren, Rotem Edgar & Udi Qimron

Authors
  1. Ido Yosef
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  2. Moran G. Goren
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  3. Rotem Edgar
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  4. Udi Qimron
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Corresponding author

Correspondence to Udi Qimron .

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Editors and Affiliations

  1. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Magnus Lundgren

  2. Department of Molecular Biology MIMS, Umeå University, Umeå, Sweden

    Emmanuelle Charpentier

  3. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

    Peter C. Fineran

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Yosef, I., Goren, M.G., Edgar, R., Qimron, U. (2015). Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_15

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_15

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2686-2

  • Online ISBN: 978-1-4939-2687-9

  • eBook Packages: Springer Protocols

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