A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells.

Détails

ID Serval
serval:BIB_A23C722A208B
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells.
Périodique
DNA repair
Auteur⸱e⸱s
Bosshard S., Duroy P.O., Mermod N.
ISSN
1568-7856 (Electronic)
ISSN-L
1568-7856
Statut éditorial
Publié
Date de publication
10/2019
Peer-reviewed
Oui
Volume
82
Pages
102691
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Résumé
CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.
Mots-clé
Animals, CHO Cells, CRISPR-Associated Protein 9/metabolism, Cricetinae, Cricetulus, DNA End-Joining Repair/genetics, Deoxyribonucleases, Type II Site-Specific/metabolism, Gene Editing, Homologous Recombination/genetics, Alternative end-joining pathways, Gene correction, Genome editing, Homologous recombination
Pubmed
Web of science
Open Access
Oui
Création de la notice
13/09/2019 18:46
Dernière modification de la notice
19/06/2020 6:21
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