Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.

ABSTRACT: An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could b

SUBMITTER: Xu W 

PROVIDER: S-EPMC9164083 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature