Genetic Modifications in Pigs for Biomedicine

Pigs have been extensively used in biomedical research and are excellent models for neurodegenerative disease. Genetic engineering in pigs was greatly enhanced by the emergence of emerging of the nuclease mediated genome editing. In current study, we investigated using TALENs to target DJ-1 genes in the porcine genome. High efficient 22% (11/50) bi-allelic targeting was obtained in the fibroblast cell colonies without foreign gene integration. Three piglets were obtained with bi-allelically mutation in DJ-1 genes via somatic cell nuclear transfer (SCNT). Western blot analysis showed that the expression of the DJ-1 protein was disrupted. Next we explored using Cripr/Cas9 to make genetic modifications by direct injection of the one cell zyogets. We developed a single blastocyst genotyping system to provide simple and rapid solution to evaluate and compare the sgRNA efficiency for a given gene locus first. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. Direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Further, by co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci (DJ-1, parkin, and PINK1 genes) in Bama miniature pigs at one step. Verified by whole genome sequencing, we concluded that the CRISPR/Cas9 system did not introduce significant off-target cleavages in the pig genome, though a high number of sgRNAs was employed.