Genetically Engineered Goats as Efficient Mammary Gland Bioreactors for Production of Recombinant Human Neutrophil Peptide 1 Using CRISPR/Cas9

Mammary gland bioreactors are promising methods for recombinant protein production. Human neutrophil peptide 1 (HNP1) exhibits antibacterial and immune-modulating properties. This study aims to establish a method to generate goats secreting HNP1 using the mammary gland as bioreactors. HNP1 transgenic goats were generated by using CRISPR/Cas9 technology to knock-in (KI) the HNP1 sequence into exon 7 of the goat β-casein (CSN2) gene under the control of the CSN2 promoter. One-cell stage embryos were cytoplasmically injected with a mixture of Cas9 mRNA, sgRNA, and a homologous plasmid including the T2A-HNP1 sequences, followed by transfer to recipient goats. A total of 22 live offspring goats were delivered, and 21 of these goats (95.45%) exhibited targeted edits at the CSN2 locus, and 2 female goats (9.09%) demonstrated successful HNP1 integration. Western blot and ELISA analyses confirmed the presence of HNP1 protein at high levels in the milk of these HNP1-positive goats, with mean concentrations of 22.10 µg/mL and 0.0092 µg/mL during the initial 60 days of lactation. Furthermore, milk from these transgenic goats exhibited notable antibacterial activity against Escherichia coli and Staphylococcus aureus, demonstrating the functionality of the expressed HNP1 protein. In conclusion, we established an efficient method for developing new transgenic goat lines as a mammary gland bioreactor, and the bioactive HNP1 protein secreted by the transgenic goat has the potential to combat microbial resistance.