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CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy

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Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2478))

Abstract

The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.

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Acknowledgments

We would like to thank members of the Rueda Lab, for useful suggestions and discussions, as well as members of the AstraZeneca Discovery Sciences team for providing purified spCas9 and dCas9 protein. We also thank LUMICKS and its Applications Scientists for assistance troubleshooting the C-Trap. The Rueda lab is funded by a core grant of the MRC-London Institute of Medical Sciences (UKRI MC-A658-5TY10), a Wellcome Trust collaborative grant (206292/C/17/Z), and a BBSRC Case Studentship (to MDN).

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Authors and Affiliations

  1. Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, UK

    Matthew D. Newton & David S. Rueda

  2. Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK

    Matthew D. Newton & David S. Rueda

  3. Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge, UK

    Benjamin J. Taylor & Maria Emanuela Cuomo

Authors
  1. Matthew D. Newton
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  2. Benjamin J. Taylor
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  3. Maria Emanuela Cuomo
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  4. David S. Rueda
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Corresponding author

Correspondence to David S. Rueda .

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Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Arne Gennerich

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Newton, M.D., Taylor, B.J., Cuomo, M.E., Rueda, D.S. (2022). CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 2478. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2229-2_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2229-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2228-5

  • Online ISBN: 978-1-0716-2229-2

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