Abstract
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 expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.
Highlights
Transplantation is an important treatment for a number of medical conditions, but the supply of human donor material remains a crucial limiting factor
We describe here an efficient method for knocking relatively large transgenes into the GGTA1 locus in pig cells using the high-fidelity FokI-dCas[9] system
Our initial experiment showed that FokI-dCas[9] used with two guide RNAs was capable of rapidly generating a sizeable proportion of biallelic GGTA1 KO pig cells, even in the absence of selection
Summary
Transplantation is an important treatment for a number of medical conditions, but the supply of human donor material remains a crucial limiting factor. The demonstrated utility of the CRISPR system to generate knock-in pigs[13,14,15] (cf knockout pigs) has not yet been applied for xenotransplantation To address these gaps, we established a FokI-dCas9-based protocol to knock-in a potentially protective transgene, encoding the anti-CD2 mAb diliximab, into the GGTA1 locus of pigs. We established a FokI-dCas9-based protocol to knock-in a potentially protective transgene, encoding the anti-CD2 mAb diliximab, into the GGTA1 locus of pigs This “kills two birds with one stone” by eliminating the gene associated with hyperacute rejection while introducing an immunomodulatory molecule
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