Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, viral infection. With objective studying TPC2's roles for first time a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem CRISPR-Cas9 edit embryos is mosaicism; thus, we studied effect microinjection timing on mosaicism. Mosaicism was greatly reduced when vitro microinjected before insemination, surgical embryo transfer (ET) performed such embryos. All fetuses piglets born following ET (i.e., F0 generation) nonmosaic biallelic KOs. The generation animals facilitates germ line transmission mutation, thereby aiding rapid efficient lines medical research agriculture.