Germline ablation achieved via CRISPR/Cas9 targeting of in bovine zygotes.

is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live knockout (KO) cattle have not been reported, and the specific role of in male cattle, or bulls, remains unexplored. This study generated KO cattle cytoplasmic microinjection of the CRISPR/Cas9 system produced bovine zygotes and evaluated the effect of elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% ( = 8/26) pregnancy rate. A 75% ( = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of KO cattle was unremarkable. Overall, this study demonstrated that the absence of in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in KO germline-ablated hosts.