Please use this identifier to cite or link to this item:
Scopus Web of Science® Altmetric
Type: Journal article
Title: Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
Author: Nottle, M.
Salvaris, E.
Fisicaro, N.
McIlfatrick, S.
Vassiliev, I.
Hawthorne, W.
O'Connell, P.
Brady, J.
Lew, A.
Cowan, P.
Citation: Scientific Reports, 2017; 7(1):8383-1-8383-8
Publisher: Nature Publishing Group
Issue Date: 2017
ISSN: 2045-2322
Statement of
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 and Peter J. Cowan
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.
Keywords: Fibroblasts
Animals, Genetically Modified
Deoxyribonucleases, Type II Site-Specific
Antibodies, Monoclonal
Reproducibility of Results
Gene Targeting
Nuclear Transfer Techniques
Gene Knock-In Techniques
CD2 Antigens
CRISPR-Associated Protein 9
Rights: © The Author(s) 2017. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit
DOI: 10.1038/s41598-017-09030-6
Grant ID:
Appears in Collections:Aurora harvest 8
Paediatrics publications

Files in This Item:
File Description SizeFormat 
hdl_109088.pdfPublished version1.39 MBAdobe PDFView/Open

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.