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Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

Author

Listed:
  • Fernando Orden Rueda

    (AstraZeneca)

  • Michal Bista

    (AstraZeneca)

  • Matthew D. Newton

    (Imperial College London)

  • Anne U. Goeppert

    (AstraZeneca)

  • M. Emanuela Cuomo

    (AstraZeneca)

  • Euan Gordon

    (AstraZeneca)

  • Felix Kröner

    (Dynamic Biosensors GmbH)

  • Jon A. Read

    (AstraZeneca)

  • Jonathan D. Wrigley

    (AstraZeneca)

  • David Rueda

    (Imperial College London)

  • Benjamin J. M. Taylor

    (AstraZeneca)

Abstract

The CRISPR–Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA–RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.

Suggested Citation

  • Fernando Orden Rueda & Michal Bista & Matthew D. Newton & Anne U. Goeppert & M. Emanuela Cuomo & Euan Gordon & Felix Kröner & Jon A. Read & Jonathan D. Wrigley & David Rueda & Benjamin J. M. Taylor, 2017. "Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease," Nature Communications, Nature, vol. 8(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01732-9
    DOI: 10.1038/s41467-017-01732-9
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    Cited by:

    1. Guanhua Xun & Zhixin Zhu & Nilmani Singh & Jingxia Lu & Piyush K. Jain & Huimin Zhao, 2024. "Harnessing noncanonical crRNA for highly efficient genome editing," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    2. Eman A. Ageely & Ramadevi Chilamkurthy & Sunit Jana & Leonora Abdullahu & Daniel O’Reilly & Philip J. Jensik & Masad J. Damha & Keith T. Gagnon, 2021. "Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    3. Amanda R. Krysler & Christopher R. Cromwell & Tommy Tu & Juan Jovel & Basil P. Hubbard, 2022. "Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences," Nature Communications, Nature, vol. 13(1), pages 1-13, December.

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