McLean ZLFermin LMAppleby SJWei JMeng FMaclean PHPerry BJBrophy BTurner PForrester-Gauntlett BWells DNSnell RGOback B2025-07-242025-07-242025-07-02McLean ZL, Fermin LM, Appleby SJ, Wei J, Meng F, Maclean PH, Perry BJ, Brophy B, Turner P, Forrester-Gauntlett B, Wells DN, Snell RG, Oback B. (2025). Morula complementation restores male germline in NANOS2 null sheep. Pnas Nexus. 4. 7.https://mro.massey.ac.nz/handle/10179/73240Current livestock breeding is slow to respond to rapidly mounting environmental pressures that threaten sustainable animal protein production. New approaches can accelerate genetic improvement by multiplying valuable embryonic, rather than adult genotypes. Chimeras, derived from complementing a sterile host with a fertile donor embryo, provide a pathway to multiply and exclusively transmit elite male germlines. We established genetically sterile hosts and optimized embryo complementation conditions to achieve absolute germline transmission in sheep. The spermatogonia-specific gene NANOS2 was disrupted in male (NANOS2+/−, NANOS2−/−) and female (NANOS2−/−) ovine fetal fibroblasts via gRNA–Cas9-mediated homology-directed repair. Targeted cell strains and wild-type controls were used to produce cloned offspring for breeding and phenotyping. Male homozygous knockout clones lacked detectable germ cells, while the somatic compartment of the testis remained intact. By contrast, male monoallelic and female biallelic targeting of NANOS2 did not affect germline development, resulting in fertile animals capable of producing fertile offspring with normal reproductive performance. The germ cell niche in NANOS2−/− hosts was most efficiently complemented by aggregating compacted morulae, rather than earlier cleavage stages, resulting in 97% blastocyst chimerization. Embryo-complemented cloned lambs from two different donor cell lines showed variable chimerism across tissues from each germ layer, including various degrees of germline colonization. A subset of germline chimeras contained normal numbers of prospermatogonia, indicating that the germline was fully restored for absolute transmission of the donor cell genotype.(c) 2025 The Author/sCC BY-NC 4.0https://creativecommons.org/licenses/by-nc/4.0/NANOS2livestockspermatogoniagermline chimeraembryo complementationJournal article10.1093/pnasnexus/pgaf2002752-6542journal-articlepgaf200