HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.
DNA Breaks, Double-Stranded
Humans
CRISPR-Cas Systems
CRISPR-Associated Protein 9
/ metabolism
DNA
/ metabolism
MRE11 Homologue Protein
/ metabolism
DNA-Binding Proteins
/ metabolism
CRISPR-Associated Proteins
/ metabolism
Gene Editing
Endonucleases
/ metabolism
Bacterial Proteins
/ metabolism
Endodeoxyribonucleases
/ metabolism
DNA End-Joining Repair
DNA Cleavage
Transcription Factors
/ metabolism
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
10 Jul 2024
10 Jul 2024
Historique:
received:
31
01
2024
accepted:
28
06
2024
medline:
11
7
2024
pubmed:
11
7
2024
entrez:
10
7
2024
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 38987539
doi: 10.1038/s41467-024-50080-y
pii: 10.1038/s41467-024-50080-y
doi:
Substances chimiques
CRISPR-Associated Protein 9
EC 3.1.-
DNA
9007-49-2
MRE11 Homologue Protein
EC 3.1.-
DNA-Binding Proteins
0
CRISPR-Associated Proteins
0
Endonucleases
EC 3.1.-
Bacterial Proteins
0
MRE11 protein, human
0
Cas12a protein
EC 3.1.-
Endodeoxyribonucleases
EC 3.1.-
RBBP8 protein, human
EC 3.1.-
Transcription Factors
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
5789Subventions
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)
ID : 310030_207588
Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)
ID : 310030_205199
Organisme : European Molecular Biology Organization (EMBO)
ID : ALTF 710-2021
Organisme : Agence Nationale de la Recherche (French National Research Agency)
ID : ANR 23-CE11-0033
Organisme : Agence Nationale de la Recherche (French National Research Agency)
ID : ANR-10-INBS-0005
Organisme : Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)
ID : R35 GM 122569
Informations de copyright
© 2024. The Author(s).
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