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

Nottle, MB; Salvaris, EJ; Fisicaro, N; McIlfatrick, S; Vassiliev, I; Hawthorne, WJ; O&#39;Connell, PJ; Brady, JL; Lew, AM; Cowan, PJ

Author(s): Nottle, MB; Salvaris, EJ; Fisicaro, N; McIlfatrick, S; Vassiliev, I; Hawthorne, WJ; O'Connell, PJ; Brady, JL; Lew, AM; Cowan, PJ;
Details: Publication Year 2017-08-16,Volume 7,Issue #1,Page 8383
Journal Title: Scientific Reports
Publication Type: Journal Article
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 alphaGal 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 alphaGal 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.
Publisher: AAAS
Research Division(s): Immunology
PubMed ID: 28814758
Publisher's Version: https://doi.org/10.1038/s41598-017-09030-6
Open Access at Publisher's Site: http://www.nature.com/articles/s41598-017-09030-6
NHMRC Grants: NHMRC/1061868, NHMRC/1037321, NHMRC/1043414, NHMRC/1080321, NHMRC/1105209,
Terms of Use/Rights Notice: http://creativecommons.org/licenses/by/4.0/
Documents: s41598-017-09030-6.pdf - (1.41MB, application/pdf)

Creation Date: 2017-08-30 02:22:13

Last Modified: 2017-09-04 01:12:57