CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

Marcela Vilarino, Sheikh Tamir Rashid, Fabian Patrik Suchy, Bret Roberts McNabb, Talitha van der Meulen, Eli J Fine, Syed Ahsan, Nurlybek Mursaliyev, Vittorio Sebastiano, Santiago Sain Diab, Mark O Huising, Hiromitsu Nakauchi, Pablo J Ross

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53 Citations (Scopus)
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Abstract

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Original languageEnglish
Article number17472
Number of pages10
JournalScientific Reports
Volume7
Issue number1
Early online date12 Dec 2017
DOIs
Publication statusPublished - 12 Dec 2017

Keywords

  • Journal Article

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