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Gene Synthesis> | … Preparation of knock-in constructs. The 3.58 kb GGTA1 knock-in backbone, including flanking NotI sites, was synthesized by GenScript (Piscataway, NJ) and cloned in the vector pUC57. It contained a MCS including sites (5′ to 3′) for FseI, AscI and EcoRI … | Get A Quote |
Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked e... More