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HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

Author

Listed:
  • Giordano Reginato

    (Università della Svizzera italiana (USI))

  • Maria Rosaria Dello Stritto

    (Università della Svizzera italiana (USI))

  • Yanbo Wang

    (Johns Hopkins University
    Stanford University School of Medicine)

  • Jingzhou Hao

    (Johns Hopkins University
    Boston Children’s Hospital)

  • Raphael Pavani

    (National Cancer Institute, NIH)

  • Michael Schmitz

    (University of Zurich, Winterthurerstrasse 190)

  • Swagata Halder

    (Università della Svizzera italiana (USI)
    Plaksha University)

  • Vincent Morin

    (Institute for Integrative Biology of the Cell (I2BC))

  • Elda Cannavo

    (Università della Svizzera italiana (USI))

  • Ilaria Ceppi

    (Università della Svizzera italiana (USI))

  • Stefan Braunshier

    (Università della Svizzera italiana (USI))

  • Ananya Acharya

    (Università della Svizzera italiana (USI))

  • Virginie Ropars

    (Institute for Integrative Biology of the Cell (I2BC))

  • Jean-Baptiste Charbonnier

    (Institute for Integrative Biology of the Cell (I2BC))

  • Martin Jinek

    (University of Zurich, Winterthurerstrasse 190)

  • Andrè Nussenzweig

    (National Cancer Institute, NIH)

  • Taekjip Ha

    (Johns Hopkins University
    Johns Hopkins University
    Boston Children’s Hospital
    Harvard Medical School)

  • Petr Cejka

    (Università della Svizzera italiana (USI))

Abstract

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Suggested Citation

  • Giordano Reginato & Maria Rosaria Dello Stritto & Yanbo Wang & Jingzhou Hao & Raphael Pavani & Michael Schmitz & Swagata Halder & Vincent Morin & Elda Cannavo & Ilaria Ceppi & Stefan Braunshier & Anan, 2024. "HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50080-y
    DOI: 10.1038/s41467-024-50080-y
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