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Internal guide RNA interactions interfere with Cas9-mediated cleavage

Author

Listed:
  • Summer B. Thyme

    (Harvard University
    Present address: Harvard University, 16 Divinity Avenue, Biolabs 1020, Cambridge, Massachusetts 02138, USA)

  • Laila Akhmetova

    (Harvard University)

  • Tessa G. Montague

    (Harvard University)

  • Eivind Valen

    (Computational Biology Unit, University of Bergen
    Sars Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgaten 55)

  • Alexander F. Schier

    (Harvard University
    Center for Brain Science, Harvard University
    Broad Institute of MIT and Harvard
    Harvard Stem Cell Institute)

Abstract

The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms11750
    DOI: 10.1038/ncomms11750
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    Cited by:

    1. Xu Feng & Ruyi Xu & Jianglan Liao & Jingyu Zhao & Baochang Zhang & Xiaoxiao Xu & Pengpeng Zhao & Xiaoning Wang & Jianyun Yao & Pengxia Wang & Xiaoxue Wang & Wenyuan Han & Qunxin She, 2024. "Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    2. 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.
    3. Fang Liang & Yu Zhang & Lin Li & Yexin Yang & Ji-Feng Fei & Yanmei Liu & Wei Qin, 2022. "SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    4. Luke Hoberecht & Pirunthan Perampalam & Aaron Lun & Jean-Philippe Fortin, 2022. "A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies," Nature Communications, Nature, vol. 13(1), pages 1-20, December.
    5. Giulia I. Corsi & Kunli Qu & Ferhat Alkan & Xiaoguang Pan & Yonglun Luo & Jan Gorodkin, 2022. "CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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