One-Step In Vitro Generation of ETV2-Null Pig Embryos

SIMPLE SUMMARY: One of the latest goals in regenerative medicine is to use pluripotent stem cells to generate whole organs in vivo through the blastocyst complementation technique. This method consists of the microinjection of pluripotent stem cells into preimplantation embryos that have been genetically modified to ablate the development of a target organ. By taking advantage of the spatiotemporal clues present in the developing embryo, pluripotent stem cells are able to colonize the empty developmental niche and create the missing organ. Combining human pluripotent stem cells with genetically engineered pig embryos, it would be possible to obtain humanized organs that could be used for transplantation, and, therefore, solve the worldwide issue of insufficient availability of transplantable organs. As endothelial cells play a critical role in xenotransplantation rejection in all organs, in this study, we optimized a protocol to generate a vascular-disabled preimplantation pig embryo using the CRISPR/Cas9 system. This protocol could be used to generate avascular embryos for blastocyst complementation experiments and work towards the generation of rejection-free humanized organs in pigs. ABSTRACT: Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.