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Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity

Author

Listed:
  • Christopher R. Cromwell

    (University of Alberta)

  • Keewon Sung

    (Seoul National University)

  • Jinho Park

    (Seoul National University)

  • Amanda R. Krysler

    (University of Alberta)

  • Juan Jovel

    (University of Alberta)

  • Seong Keun Kim

    (Seoul National University)

  • Basil P. Hubbard

    (University of Alberta)

Abstract

Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.

Suggested Citation

  • Christopher R. Cromwell & Keewon Sung & Jinho Park & Amanda R. Krysler & Juan Jovel & Seong Keun Kim & Basil P. Hubbard, 2018. "Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-03927-0
    DOI: 10.1038/s41467-018-03927-0
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    Cited by:

    1. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. 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.
    3. 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.
    4. 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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