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.