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Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Author

Listed:
  • Kazuto Yoshimi

    (University of Tokyo
    University of Tokyo)

  • Kohei Takeshita

    (Advanced Photon Technology Division)

  • Noriyuki Kodera

    (Kanazawa University, Kakuma-machi)

  • Satomi Shibumura

    (C4U Corporation)

  • Yuko Yamauchi

    (University of Tokyo)

  • Mine Omatsu

    (Advanced Photon Technology Division
    University of Hyogo)

  • Kenichi Umeda

    (Kanazawa University, Kakuma-machi)

  • Yayoi Kunihiro

    (C4U Corporation)

  • Masaki Yamamoto

    (Advanced Photon Technology Division
    University of Hyogo)

  • Tomoji Mashimo

    (University of Tokyo
    University of Tokyo)

Abstract

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

Suggested Citation

  • Kazuto Yoshimi & Kohei Takeshita & Noriyuki Kodera & Satomi Shibumura & Yuko Yamauchi & Mine Omatsu & Kenichi Umeda & Yayoi Kunihiro & Masaki Yamamoto & Tomoji Mashimo, 2022. "Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-32618-0
    DOI: 10.1038/s41467-022-32618-0
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    References listed on IDEAS

    as
    1. Kazuto Yoshimi & Yayoi Kunihiro & Takehito Kaneko & Hitoshi Nagahora & Birger Voigt & Tomoji Mashimo, 2016. "ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes," Nature Communications, Nature, vol. 7(1), pages 1-10, April.
    2. Kirill A. Datsenko & Ksenia Pougach & Anton Tikhonov & Barry L. Wanner & Konstantin Severinov & Ekaterina Semenova, 2012. "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system," Nature Communications, Nature, vol. 3(1), pages 1-7, January.
    3. Anna A. Shiriaeva & Ekaterina Savitskaya & Kirill A. Datsenko & Irina O. Vvedenskaya & Iana Fedorova & Natalia Morozova & Anastasia Metlitskaya & Anton Sabantsev & Bryce E. Nickels & Konstantin Severi, 2019. "Detection of spacer precursors formed in vivo during primed CRISPR adaptation," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    4. Hongtu Zhao & Gang Sheng & Jiuyu Wang & Min Wang & Gabor Bunkoczi & Weimin Gong & Zhiyi Wei & Yanli Wang, 2014. "Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli," Nature, Nature, vol. 515(7525), pages 147-150, November.
    5. Blake Wiedenheft & Gabriel C. Lander & Kaihong Zhou & Matthijs M. Jore & Stan J. J. Brouns & John van der Oost & Jennifer A. Doudna & Eva Nogales, 2011. "Structures of the RNA-guided surveillance complex from a bacterial immune system," Nature, Nature, vol. 477(7365), pages 486-489, September.
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    Cited by:

    1. Anna Zimmermann & Julian E. Prieto-Vivas & Charlotte Cautereels & Anton Gorkovskiy & Jan Steensels & Yves Peer & Kevin J. Verstrepen, 2023. "A Cas3-base editing tool for targetable in vivo mutagenesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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