CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

Marcela Vilarino, Sheikh Tamir Rashid, Fabian Patrik Suchy, Bret McNabb, Talitha Van Der Meulen, Eli J. Fine, Syed Ahsan, Nurlybek Mursaliyev, Vittorio Sebastiano, Santiago S Diab, Mark O. Huising, Hiromitsu Nakauchi, Pablo J. Ross

Research output: Contribution to journalArticle

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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 (US)
Article number17472
JournalScientific Reports
Volume7
Issue number1
DOIs
StatePublished - Dec 1 2017

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Clustered Regularly Interspaced Short Palindromic Repeats
Microinjections
Oocytes
Pancreas
Sheep
Pluripotent Stem Cells
Zygote
Blastocyst
Metaphase
Fetus
Mutation
Mosaicism
Regenerative Medicine
Gene Silencing
Genes
Embryonic Structures

ASJC Scopus subject areas

  • General

Cite this

Vilarino, M., Rashid, S. T., Suchy, F. P., McNabb, B., Van Der Meulen, T., Fine, E. J., ... Ross, P. J. (2017). CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Scientific Reports, 7(1), [17472]. https://doi.org/10.1038/s41598-017-17805-0

CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. / Vilarino, Marcela; Rashid, Sheikh Tamir; Suchy, Fabian Patrik; McNabb, Bret; Van Der Meulen, Talitha; Fine, Eli J.; Ahsan, Syed; Mursaliyev, Nurlybek; Sebastiano, Vittorio; Diab, Santiago S; Huising, Mark O.; Nakauchi, Hiromitsu; Ross, Pablo J.

In: Scientific Reports, Vol. 7, No. 1, 17472, 01.12.2017.

Research output: Contribution to journalArticle

Vilarino, M, Rashid, ST, Suchy, FP, McNabb, B, Van Der Meulen, T, Fine, EJ, Ahsan, S, Mursaliyev, N, Sebastiano, V, Diab, SS, Huising, MO, Nakauchi, H & Ross, PJ 2017, 'CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep', Scientific Reports, vol. 7, no. 1, 17472. https://doi.org/10.1038/s41598-017-17805-0
Vilarino, Marcela ; Rashid, Sheikh Tamir ; Suchy, Fabian Patrik ; McNabb, Bret ; Van Der Meulen, Talitha ; Fine, Eli J. ; Ahsan, Syed ; Mursaliyev, Nurlybek ; Sebastiano, Vittorio ; Diab, Santiago S ; Huising, Mark O. ; Nakauchi, Hiromitsu ; Ross, Pablo J. / CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. In: Scientific Reports. 2017 ; Vol. 7, No. 1.
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