AUTHOR=Mueller Maci L. , McNabb Bret R. , Owen Joseph R. , Hennig Sadie L. , Ledesma Alba V. , Angove Mitchell L. , Conley Alan J. , Ross Pablo J. , Van Eenennaam Alison L. 

TITLE=Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes

JOURNAL=Frontiers in Genome Editing

VOLUME=5

YEAR=2023

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2023.1321243

DOI=10.3389/fgeed.2023.1321243

ISSN=2673-3439

ABSTRACT=<p><italic>NANOS3</italic> 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 <italic>NANOS3</italic> knockout (KO) cattle have not been reported, and the specific role of <italic>NANOS3</italic> in male cattle, or bulls, remains unexplored. This study generated <italic>NANOS3</italic> KO cattle <italic>via</italic> cytoplasmic microinjection of the CRISPR/Cas9 system <italic>in vitro</italic> produced bovine zygotes and evaluated the effect of <italic>NANOS3</italic> 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 <italic>NANOS3</italic> KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (<italic>n</italic> = 8/26) pregnancy rate. A 75% (<italic>n</italic> = 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 <italic>NANOS3</italic> 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 <italic>NANOS3</italic> KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, <italic>NANOS3</italic> 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, <italic>NANOS3</italic> KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in <italic>NANOS3</italic> KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of <italic>NANOS3</italic> KO cattle was unremarkable. Overall, this study demonstrated that the absence of <italic>NANOS3</italic> in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of <italic>NANOS3</italic> KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of <italic>NANOS3</italic> function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in <italic>NANOS3</italic> KO germline-ablated hosts.</p>