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Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

Author

Listed:
  • Stephan Riesenberg

    (Max Planck Institute for Evolutionary Anthropology)

  • Nelly Helmbrecht

    (Max Planck Institute for Evolutionary Anthropology)

  • Philipp Kanis

    (Max Planck Institute for Evolutionary Anthropology)

  • Tomislav Maricic

    (Max Planck Institute for Evolutionary Anthropology)

  • Svante Pääbo

    (Max Planck Institute for Evolutionary Anthropology
    Okinawa Institute of Science and Technology)

Abstract

The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.

Suggested Citation

  • Stephan Riesenberg & Nelly Helmbrecht & Philipp Kanis & Tomislav Maricic & Svante Pääbo, 2022. "Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28137-7
    DOI: 10.1038/s41467-022-28137-7
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    References listed on IDEAS

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    1. C. D. Richardson & G. J. Ray & N. L. Bray & J. E. Corn, 2016. "Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes," Nature Communications, Nature, vol. 7(1), pages 1-7, November.
    2. Summer B. Thyme & Laila Akhmetova & Tessa G. Montague & Eivind Valen & Alexander F. Schier, 2016. "Internal guide RNA interactions interfere with Cas9-mediated cleavage," Nature Communications, Nature, vol. 7(1), pages 1-7, September.
    3. Daqi Wang & Chengdong Zhang & Bei Wang & Bin Li & Qiang Wang & Dong Liu & Hongyan Wang & Yan Zhou & Leming Shi & Feng Lan & Yongming Wang, 2019. "Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
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    1. Dmitrii Degtev & Jack Bravo & Aikaterini Emmanouilidi & Aleksandar Zdravković & Oi Kuan Choong & Julia Liz Touza & Niklas Selfjord & Isabel Weisheit & Margherita Francescatto & Pinar Akcakaya & Michel, 2024. "Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles," Nature Communications, Nature, vol. 15(1), pages 1-15, December.

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