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CRISPR pp 133–145Cite as

Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS)

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

Abstract

Mass spectrometry is a powerful tool for characterizing RNA. Here we describe a method for the identification and characterisation of crRNA using liquid chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS). The direct purification of crRNA from the Cascade-crRNA complex was performed using denaturing ion pair reverse phase chromatography. Following purification of the crRNA, the intact mass was determined by LC ESI MS. Using this approach, a significant reduction in metal ion adduct formation of the crRNA was observed. In addition, RNase mapping of the crRNA was performed using RNase digestion in conjunction with liquid chromatography tandem MS analysis. Using the intact mass of the crRNA, in conjunction with RNase mapping experiments enabled the identification and characterisation of the crRNA, providing further insight into crRNA processing in a number of type I CRISPR-Cas systems.

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Acknowledgments

MJD acknowledges funding support from the Engineering and Physical Sciences Research Council UK and the Biotechnology and Biological Sciences Research Council Research Council UK.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin St, Sheffield, UK

    Sakharam P. Waghmare, Alison O. Nwokeoji & Mark J. Dickman

  2. Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow, UK

    Sakharam P. Waghmare

Authors
  1. Sakharam P. Waghmare
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  2. Alison O. Nwokeoji
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  3. Mark J. Dickman
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Corresponding author

Correspondence to Mark J. Dickman .

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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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Waghmare, S.P., Nwokeoji, A.O., Dickman, M.J. (2015). Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS). 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_8

  • 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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