Rabbits are valuable for generating human disease models due to their physiological similarities to humans. However, the generation of germline-competent chimeras with genome edits remains challenging. This limitation is largely due to the lack of pluripotent stem cells (PSCs) with strong embryo-colonization potential, a feature well-established in mice and rats, but not in any other mammalian species.

We recently identified three genes–KLF2, ERAS, and PRMT6–whose overexpression in rabbit induced pluripotent stem cells (iPSCs) markedly enhances their ability to colonize host preimplantation embryos. iPSCs engineered to overexpress these genes and cultured in an optimized medium containing Activin A, leukemia inhibitor factor (LIF), Vitamin C, and inhibitors of protein kinase C (PKC) and Tankyrases exhibited molecular and epigenetic hallmarks of naïve pluripotency. These included high CD75 expression and X chromosome reactivation. To enable temporal control of transgene expression, we established inducible KLF2-ERAS-PRMT6-expressing iPSC lines (KEPi) using the ProteoTuner™ system. These cells showed upregulation of naïve markers, and demonstrated robust embryo-colonization capacity. Notably, CD75high-sorted KEPi cells achieved complete colonization of host embryos and gave rise to viable chimeric rabbits with confirmed germline transmission.

To overcome reliance on exogenous transgenes and enable precise modulation of endogenous gene networks, we are now employing CRISPR-mediated activation (CRISPRa). We established a novel rabbit iPSC line (CRISPRai#5) harboring an inducible dCas9 fused to a transcriptional activation domain, allowing targeted activation of KLF2, ERAS, and PRMT6 . Preliminary experiments demonstrated successful colonization of rabbit preimplantation embryos. Ongoing studies aim to determine whether these embryos can give rise to germline chimeras following transfer into surrogate mothers.

This work represents a significant advance toward establishing robust naïve pluripotency in non-rodent species and enhancing their utility for human disease modeling.