Targeting of cholera toxin A () gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.

Background And Purpose: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene () for inhibiting CT toxin production in .

Experimental Approach: An engineered ZFN was designed to target the catalytic site of the gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and plasmid and transformed to Top10 and . The efficiency of ZFN was evaluated by colony counting.

Findings/results: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed . The gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of with vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay.

Conclusions And Implications: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.