One-step generation of modified cattle and sheep from spermatid-like haploid stem cells

Genome-modified ruminants, such as cattle and sheep, are important germplasm for agricultural applications. Haploid androgenetic stem cells (haSCs) can support the full-term development of embryos upon intracytoplasmic haSCs injection (iCHI) into intact oocytes. Thus, haSCs are invaluable resources for studying economic traits and significantly impact livestock breeding; however, ruminant haSCs have yet to be obtained. Here, we report the derivation of cattle and sheep haSCs using a recombinant FACE medium. These cells display characteristics of formative-state pluripotency and can differentiate into three germ layers, both in vitro and in vivo. Interestingly, the ectopic expression of protamine in haSCs converts their nuclei into a spermatid-like structure and synergistic enhancement of full-term development of iCHI embryos, named as Pro-iCHI approach. Furthermore, gene-modified cattle and sheep can be efficiently produced through the combinatorial use of the CRISPR and Pro-iCHI approach. Overall, Pro-iCHI provides an easy-to-manipulate agrobiotechnology tool for rapidly producing genetically modified livestock.