One-step in vitro generation of ETV2-Null pig embryos

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 c...

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Autores: Moya-Jódar, M. (Marta)|||/items/45efbc40-bb93-44a3-9504-667e025846f2, Coppiello, G. (Giulia)|||/items/d1d63554-537e-458e-8f86-02ae9264fe14, Rodriguez-Madoz, J.R. (Juan Roberto)|||/items/9c21019b-aeba-4cdc-b78e-178a176da535, Abizanda-Sarasa, G. (Gloria)|||/items/1e76a0e3-32d0-424e-b58a-d9ddf7a82ef7, Barlabé-Ginesta, P. (Paula)|||/items/9060368a-beba-4ce3-b04c-35da49542053, Vilas-Zornoza, A. (Amaia)|||/items/7435672d-e471-4409-8325-006fef27fb12, Ullate-Agote, A. (Asier)|||/items/af0561e9-aebe-4e94-9cb1-6bb3aa8bf975, Luongo, C. (Chiara)|||/items/7e44fc11-311c-4793-a923-26ed2a354a88, Rodríguez-Tobón, E. (Ernesto)|||/items/2cec1a67-1135-4f0a-8252-fb19c99a037c, Navarro-Serna, S. (Sergio)|||/items/6e7a562e-2c20-4258-b33b-bd98ed24815a, París-Oller, E. (Evelyne)|||/items/e29dc986-47b7-427b-99fa-6d9e4c3bc48b, Oficialdegui, M. (María)|||/items/a0992453-91fc-424d-a871-26976f7cedb9, Carvajal-Vergara, X. (Xonia)|||/items/5ea998ea-511c-410a-9686-fb4da1db3b78, Ordovás, L. (Laura)|||/items/ffb706a8-de41-4c2e-a0cf-fb2fe6d160d6, Prosper-Cardoso, F. (Felipe)|||/items/3d1b0b82-06c3-4e63-8280-e903dc4dc0c1, García-Vázquez, F. A. (Francisco Alberto)|||/items/b3f34ebd-eafe-4e8f-9ae0-11c318c1515b, Aranguren-López, X. (Xabier)|||/items/217519c3-f392-48cb-b4c9-87d4e2fd0570
Tipo de recurso: artículo
Fecha de publicación:2022
País:España
Institución:Universidad de Navarra
Repositorio:Dadun. Depósito Académico Digital de la Universidad de Navarra
Idioma:inglés
OAI Identifier:oai:dadun.unav.edu:10171/116539
Acceso en línea:https://hdl.handle.net/10171/116539
Access Level:acceso abierto
Palabra clave:CRISPR/Cas9
ETV2
Gene editing
Porcine embryos
Vascular development
Descripción
Sumario: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.

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