Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells

In rodents, injection of haploid androgenetic embryonic stem cells (haES cells) into intact oocytes enables full-term development of offspring. The value of this method in research and genome engineering has not been replicated in ruminants because ruminant haES cells are yet to be obtained. Here we report the derivation of cow and sheep haES cells and their application to generate offspring by a method we call intracytoplasmic haES cell injection (iCHI), in analogy with intracytoplasmic sperm injection. Ruminant haES cells display characteristics of formative-state pluripotency and differentiate into the three germ layers both in vitro and in vivo. Ectopic expression of protamine in haES cells converts their nuclei into a spermatid-like structure and improves full-term development of iCHI embryos, a method we call protamine iCHI (Pro-iCHI). We also combine Pro-iCHI and prime editing to generate gene-modified cows and sheep. Overall, Pro-iCHI provides a promising approach for production of genetically modified livestock.