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.
Highlights
One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs)
In the future, combining gene editing with CRISPR/Cas[9] and PSCs complementation could result in a powerful approach for human organ generation
Development to the blastocyst stage was higher in control (31.5%, 87/276) and metaphase II (MII)-microinjected embryos (27.1%, 100/369) versus zygote-microinjected embryos (16.1%, 53/329; p < 0.05), irrespective of embryo production method (PA or in vitro fertilized (IVF)) (Fig. 1b; Supplementary information, Table S1)
Summary
One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). We report here the creation of pancreatogenesisdisabled sheep by oocyte microinjection of CRISPR/Cas[9] targeting PDX1, a critical gene for pancreas development. 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/Cas[9] and PSCs complementation could result in a powerful approach for human organ generation. As a proof of principle, the possibility for intra- and interspecies blastocyst complementation has been demonstrated using rodent models[3,4,5] These results raised the possibility of generating functional human tissues and organs within an animal species (hosts) with similar anatomy, size, and physiology to humans[6]. We provide an effective and efficient approach for the production of gene-edited sheep that could be used for patient-specific human organ generation
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