One-Step In Vitro Generation of ETV2-Null Pig Embryos
Marta Moya-Jódar,
Giulia Coppiello,
Juan Roberto Rodríguez-Madoz,
Gloria Abizanda,
Paula Barlabé,
Amaia Vilas-Zornoza,
Asier Ullate-Agote,
Chiara Luongo,
Ernesto Rodríguez-Tobón,
Sergio Navarro-Serna,
Evelyne París-Oller,
Maria Oficialdegui,
Xonia Carvajal-Vergara,
Laura Ordovás,
Felipe Prósper,
Francisco Alberto García-Vázquez,
Xabier L. Aranguren
Affiliations
Marta Moya-Jódar
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Giulia Coppiello
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Juan Roberto Rodríguez-Madoz
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Gloria Abizanda
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Paula Barlabé
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Amaia Vilas-Zornoza
Advanced Genomics Laboratory, Program of Hemato-Oncology, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain
Asier Ullate-Agote
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Chiara Luongo
Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain
Ernesto Rodríguez-Tobón
Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain
Sergio Navarro-Serna
Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain
Evelyne París-Oller
Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain
Maria Oficialdegui
Granja Los Alecos, 31395 Barásoain, Spain
Xonia Carvajal-Vergara
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Laura Ordovás
Aragon Agency for Research and Development (ARAID), 50018 Zaragoza, Spain
Felipe Prósper
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
Francisco Alberto García-Vázquez
Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain
Xabier L. Aranguren
Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
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.