ZeoR: A Candidate for Assays Testing Enzymatic Activities and Off-Target Effects of Gene-Editing Enzymes

The target lengths of gene-editing enzymes vary from approximately 20 to 80 nucleotides. The ideal reporter genes should be able to tolerate the insertion of target sequences to differentiate the phenotypic changes between in-frame and frame shift mutations for evaluating the enzymatic activities and off-target effects of gene-editing enzymes such as zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats-associated system. This study demonstrated that the zeocin resistance gene (ZeoR) tolerated the insertion of long targets for a variety of tests using a strategy of inserting tandem repeats of NNT. Four genes such as CCR5, AR, HPV 16E7 early gene, and EGFR applications of ZeoR in quantitatively analyzing the off-targets of CRISPR were successful. The data indicated that evaluating the enzymatic activities and off-target effects with assays employing ZeoR had great potential in facilitating the application of gene editing to human gene therapy.