Evaluation Of Gene Editing Enzymes’ Acting Based On Zeocin R Reporter Gene

Objective: To develop new assays for the evaluation of the enzymatic activities and off-target effects of gene editing enzymes.Methods: Bleomycin(zeocin) resistance gene(Zeo R) was chosen as the target of a new system for the evaluation of the enzymatic activities and off-target effects of gene editing enzymes. A prokaryotic vector possessing double antibiotic activities, Amp R-Zeo R was first constructed. An enzyme site of Bae I that is compatible to Golden gate cloning was added between the initiator triplet of ATG and the second codon of GCC. A vector with resistance to G418/Kana-Zeocin was constructed for applications in eukaryotic cells, the eukaryotic and prokaryotic vectors share their properties of being digested by Bae I. To test if Zeo R can be used in gene editing related studies, a rescue assay was performed in E. coli by yielding double stranded break on the Zeo R gene and co-transformed with a homologous oligo.Results: Successfully developed double antibiotic vector with Zeo R and Amp R, and observed the ideal selection of either maintained or disappeared drug resistance following the insertion of either in-frame or frameshift fragments between the first and the second triplets of the Zeo R gene. Additionally, drug resistance also lost after insertion with nonsense mutation. Finally, both the lost drug resistance either by inframe or nonsense mutations had been rescued by gene editing in E. coli.Conclusion: This study comprehensively characterized the capacity of Zeo R gene in response to inserted fragments at the site between the first and the second triplets in the newly developed double antibiotic vector. The zeocin gene provided an ideal on and off switch in responding to inserts inframe and the frameshift fragments or with nonsense mutation. The zeo R assay can be used in the evaluation of enzymatic activity and off-target effects of gene editing enzymes.