Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells.
Fiche publication
Date publication
février 2023
Journal
Genes
Auteurs
Membres identifiés du Cancéropôle Est :
Dr HERAULT Yann
Tous les auteurs :
Erbs V, Lorentz R, Eisenman B, Schaeffer L, Luppi L, Lindner L, Hérault Y, Pavlovic G, Wattenhofer-Donzé M, Birling MC
Lien Pubmed
Résumé
The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for 'à la carte' mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5' and 3' PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.
Mots clés
CRISPR/Cas9, double-strand break, embryonic stem cells, gene targeting, genome editing, homologous recombination, reproducibility
Référence
Genes (Basel). 2023 02 3;14(2):