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Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9

Sci Rep. 2017-08; 
NottleMark B, SalvarisEvelyn J, FisicaroNella, McIlfatrickStephen, VassilievIvan, HawthorneWayne J, O'ConnellPhilip J, BradyJamie L, LewAndrew M, CowanPet
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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

摘要

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

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