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

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-genera...

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Autores principales:	Mark B. Nottle, Evelyn J. Salvaris, Nella Fisicaro, Stephen McIlfatrick, Ivan Vassiliev, Wayne J. Hawthorne, Philip J. O’Connell, Jamie L. Brady, Andrew M. Lew, Peter J. Cowan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/13fe765c6d834f9facf6f7bbc3879a6c
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spelling	oai:doaj.org-article:13fe765c6d834f9facf6f7bbc3879a6c2021-12-02T15:05:36ZTargeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas910.1038/s41598-017-09030-62045-2322https://doaj.org/article/13fe765c6d834f9facf6f7bbc3879a6c2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09030-6https://doaj.org/toc/2045-2322Abstract 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.Mark B. NottleEvelyn J. SalvarisNella FisicaroStephen McIlfatrickIvan VassilievWayne J. HawthornePhilip J. O’ConnellJamie L. BradyAndrew M. LewPeter J. CowanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mark B. Nottle Evelyn J. Salvaris Nella Fisicaro Stephen McIlfatrick Ivan Vassiliev Wayne J. Hawthorne Philip J. O’Connell Jamie L. Brady Andrew M. Lew Peter J. Cowan Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
description	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.
format	article
author	Mark B. Nottle Evelyn J. Salvaris Nella Fisicaro Stephen McIlfatrick Ivan Vassiliev Wayne J. Hawthorne Philip J. O’Connell Jamie L. Brady Andrew M. Lew Peter J. Cowan
author_facet	Mark B. Nottle Evelyn J. Salvaris Nella Fisicaro Stephen McIlfatrick Ivan Vassiliev Wayne J. Hawthorne Philip J. O’Connell Jamie L. Brady Andrew M. Lew Peter J. Cowan
author_sort	Mark B. Nottle
title	Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
title_short	Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
title_full	Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
title_fullStr	Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
title_full_unstemmed	Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
title_sort	targeted insertion of an anti-cd2 monoclonal antibody transgene into the ggta1 locus in pigs using foki-dcas9
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/13fe765c6d834f9facf6f7bbc3879a6c
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Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9

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