Targeted Editing Of Goat Beta-Lactoglobulin(BLG) Gene With Modular-Assembly Zinc Finger Nucleases

Beta-lactoglobulin(BLG) is a major protein in the milk (56-60%of total cow whey proteins) and is considered to be a dominant allergen. Goat milk containing low-level milk allergy will obviously enjoy a much wider use in the food industry. ZFNs that knock out BLG may be used for transgenic goat breeding to produce goat milk with lower BLG content.Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. BLG is the only one ruminant genes that have been successfully edited with ZFNs generated by Sigma-Aldrich Biotechnology.Here we optimized the protocol to construct ZFNs using overlapping PCR and synthesized and tested6pairs of three-or four-finger ZFNs to target one site in goat BLG gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated72h later for genome editing efficiency assay. The results demonstrated that ZFNs showed considerable activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.