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CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

  • Vilarino, M
  • Rashid, ST
  • Suchy, FP
  • McNabb, BR
  • Van Der Meulen, T
  • Fine, EJ
  • Ahsan, S
  • Mursaliyev, N
  • Sebastiano, V
  • Diab, SS
  • Huising, MO
  • Nakauchi, H
  • Ross, PJ
Publication Date
Dec 01, 2017
eScholarship - University of California
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© 2017 The Author(s). 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.

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