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Structural insights into DNA cleavage activation of CRISPR-Cas9 system

Author

Listed:
  • Cong Huai

    (School of Life Sciences, Fudan University)

  • Gan Li

    (School of Life Sciences, Fudan University)

  • Ruijie Yao

    (School of Life Sciences, Fudan University)

  • Yingyi Zhang

    (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Mi Cao

    (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Liangliang Kong

    (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Chenqiang Jia

    (School of Life Sciences, Fudan University)

  • Hui Yuan

    (School of Life Sciences, Fudan University)

  • Hongyan Chen

    (School of Life Sciences, Fudan University)

  • Daru Lu

    (School of Life Sciences, Fudan University)

  • Qiang Huang

    (School of Life Sciences, Fudan University)

Abstract

CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.

Suggested Citation

  • Cong Huai & Gan Li & Ruijie Yao & Yingyi Zhang & Mi Cao & Liangliang Kong & Chenqiang Jia & Hui Yuan & Hongyan Chen & Daru Lu & Qiang Huang, 2017. "Structural insights into DNA cleavage activation of CRISPR-Cas9 system," Nature Communications, Nature, vol. 8(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01496-2
    DOI: 10.1038/s41467-017-01496-2
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    Cited by:

    1. 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.
    2. 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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