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

doi:10.1038/s41598-017-17805-0

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

Centre for Stem Cells & Regenerative Medicine

Research output: Contribution to journal › Article › peer-review

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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 language	English
Article number	17472
Number of pages	10
Journal	Scientific Reports
Volume	7
Issue number	1
Early online date	12 Dec 2017
DOIs	https://doi.org/10.1038/s41598-017-17805-0
Publication status	Published - 12 Dec 2017

Keywords

Journal Article

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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CRISPR Cas9 microinjection_VILARINO_Publishedonline12December2017_GOLD CORRECTED PROOFProof, 6.03 MBLicence: CC BY

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title = "CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep",

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.",

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AU - Vilarino, Marcela

AU - Rashid, Sheikh Tamir

AU - Suchy, Fabian Patrik

AU - McNabb, Bret Roberts

AU - van der Meulen, Talitha

AU - Fine, Eli J

AU - Ahsan, Syed

AU - Mursaliyev, Nurlybek

AU - Sebastiano, Vittorio

AU - Diab, Santiago Sain

AU - Huising, Mark O

AU - Nakauchi, Hiromitsu

AU - Ross, Pablo J

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N2 - 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.

AB - 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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CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

Abstract

Keywords

UN SDGs

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