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CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

Author

Listed:
  • Matthew A. Coelho

    (Discovery Sciences, R&D, AstraZeneca
    Wellcome Sanger Institute)

  • Etienne Braekeleer

    (Discovery Sciences, R&D, AstraZeneca)

  • Mike Firth

    (Discovery Sciences, R&D, AstraZeneca)

  • Michal Bista

    (Discovery Sciences, R&D, AstraZeneca)

  • Sebastian Lukasiak

    (Discovery Sciences, R&D, AstraZeneca)

  • Maria Emanuela Cuomo

    (Oncology R&D, AstraZeneca)

  • Benjamin J. M. Taylor

    (Discovery Sciences, R&D, AstraZeneca)

Abstract

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

Suggested Citation

  • Matthew A. Coelho & Etienne Braekeleer & Mike Firth & Michal Bista & Sebastian Lukasiak & Maria Emanuela Cuomo & Benjamin J. M. Taylor, 2020. "CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17952-5
    DOI: 10.1038/s41467-020-17952-5
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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. Qinchang Chen & Guohui Chuai & Haihang Zhang & Jin Tang & Liwen Duan & Huan Guan & Wenhui Li & Wannian Li & Jiaying Wen & Erwei Zuo & Qing Zhang & Qi Liu, 2023. "Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints," Nature Communications, Nature, vol. 14(1), pages 1-17, December.

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