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

Giordano Reginato, Maria Rosaria Dello Stritto, Yanbo Wang, Jingzhou Hao, Raphael Pavani, Michael Schmitz, Swagata Halder, Vincent Morin, Elda Cannavo, Ilaria Ceppi, Stefan Braunshier, Ananya Acharya, Virginie Ropars, Jean-Baptiste Charbonnier, Martin Jinek, Andrè Nussenzweig, Taekjip Ha and Petr Cejka ()
Additional contact information
Giordano Reginato: Università della Svizzera italiana (USI)
Maria Rosaria Dello Stritto: Università della Svizzera italiana (USI)
Yanbo Wang: Johns Hopkins University
Jingzhou Hao: Johns Hopkins University
Raphael Pavani: National Cancer Institute, NIH
Michael Schmitz: University of Zurich, Winterthurerstrasse 190
Swagata Halder: Università della Svizzera italiana (USI)
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
Petr Cejka: Università della Svizzera italiana (USI)

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: 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.

Date: 2024
References: View references in EconPapers View complete reference list from CitEc
Citations:

Downloads: (external link)
https://www.nature.com/articles/s41467-024-50080-y Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50080-y

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-024-50080-y

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().