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Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

Author

Listed:
  • Youngbin Lim

    (Seoul National University)

  • So Young Bak

    (Seoul National University)

  • Keewon Sung

    (Seoul National University)

  • Euihwan Jeong

    (Seoul National University)

  • Seung Hwan Lee

    (Center for Genome Engineering, Institute for Basic Science)

  • Jin-Soo Kim

    (Seoul National University
    Center for Genome Engineering, Institute for Basic Science)

  • Sangsu Bae

    (Seoul National University
    Center for Genome Engineering, Institute for Basic Science
    Present address: Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea)

  • Seong Keun Kim

    (Seoul National University
    Seoul National University)

Abstract

The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA–DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

Suggested Citation

  • Youngbin Lim & So Young Bak & Keewon Sung & Euihwan Jeong & Seung Hwan Lee & Jin-Soo Kim & Sangsu Bae & Seong Keun Kim, 2016. "Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease," Nature Communications, Nature, vol. 7(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms13350
    DOI: 10.1038/ncomms13350
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    Cited by:

    1. Péter István Kulcsár & András Tálas & Zoltán Ligeti & Eszter Tóth & Zsófia Rakvács & Zsuzsa Bartos & Sarah Laura Krausz & Ágnes Welker & Vanessza Laura Végi & Krisztina Huszár & Ervin Welker, 2023. "A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    2. Yang Liu & Filipe Pinto & Xinyi Wan & Zhugen Yang & Shuguang Peng & Mengxi Li & Jonathan M. Cooper & Zhen Xie & Christopher E. French & Baojun Wang, 2022. "Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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