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- Title
CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.
- Authors
Vilarino, Marcela; Rashid, Sheikh Tamir; Suchy, Fabian Patrik; McNabb, Bret Roberts; van der Meulen, Talitha; Fine, Eli J.; Ahsan, Syed Daniyal; Mursaliyev, Nurlybek; Sebastiano, Vittorio; Diab, Santiago Sain; Huising, Mark O.; Nakauchi, Hiromitsu; Ross, Pablo J.
- 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.
- Publication
Scientific Reports, 2017, Vol 7, Issue 1, p1
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-017-17805-0