Spermatogonial Stem Cell Editing and Surrogate Sires: Strategies, Bottlenecks and a Roadmap for Porcine Male Germline Editing

The porcine male germline is the route by which a validated genome edit becomes a heritable trait, and where most large-animal editing programmes slow down. Cloning and zygote microinjection remain the standard routes to an edited founder, but cloning is inefficient and zygote editing often yields mosaicism that breeding must later resolve. Spermatogonial stem cells (SSCs) offer a different order of operations: the edit is completed and verified in culture, and the germline is restored by transplantation into a recipient whose own spermatogenesis has been ablated, a design often called a surrogate sire. This review follows the three links of that chain. Enrichment by differential plating, light-scatter sorting, STA-PUT or SSEA4 selection now delivers highly enriched undifferentiated spermatogonia, and defined growth factors, Sertoli cell feeders, extracellular matrix, small molecules or bioreactors sustain expansion for weeks to months, although precise single-nucleotide replacement remains inefficient. NANOS2 knockout is the only recipient strategy shown in pigs to combine male-specific germline ablation, an intact somatic niche and fertility of the genotypes needed to propagate the line. We add a cross-link analysis of where the chain fails and a roadmap with explicit pass criteria, centred on donor-derived sperm as a fraction of total sperm.