Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

ABSTRACT: We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivatio

SUBMITTER: Killian T 

PROVIDER: S-EPMC5684134 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature