One-step generation of CRISPR/Cas9-mediated gene-edited porcine zygotes and fetuses
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One-step generation of CRISPR/Cas9-mediated gene-edited porcine zygotes and fetuses


Pigs are good candidates for studying human diseases because of their similarity to humans in physiology, organ size, and genetics. As a result of recent advances of assisted reproductive technologies (ART) along with CRISPR/Cas9 gene-editing system, efficient generation of knock-out (KO) livestock animals became easier than before. However, there is no robust protocol that can be used to generate porcine embryos because of the variabilities compared to other livestock species. In addition, inefficiencies related to the CRISPR/Cas9-mediated genome editing in porcine embryos limit the production of founder animals.This study first demonstrated that the effects of different exposure time of hormone treatments on porcine oocyte maturation and embryo development to the blastocyst stage and the effects of three cytokines, FGF2, LIF, and IGF1 (FLI) supplemented to a defined maturation media on oocyte maturation and embryo development. Since microinjection is the most used method for introducing CRISPR/Cas9 complex in embryos, we optimized the condition for efficiently disrupting pig neurogenin 3 (NGN3) gene by testing three guide RNAs (gRNAs) targeting different regions of exon 2 in the gene, comparing Cas9 mRNA and protein, and comparing three types of gRNAs, in vitro transcribed, two-part cr/tracr gRNA, and synthetic gRNA. Electroporation has been suggested as an alternative method to deliver CRISPR/Cas9 components with ease although mosaicism and off-target effects induced by CRISPR/Cas9 system are not clear. We optimized the electroporation parameter to efficiently introduce CRISPR/Cas9 targeting NGN3 in pig embryos by testing different concentrations of gRNA and Cas9 protein and subsequently determined mosaicism and off-target effects caused by electroporation procedure using next generation sequencing (PacBio).

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