Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells.

ABSTRACT: 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 lo

SUBMITTER: Erbs V 

PROVIDER: S-EPMC9957269 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature