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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| Format: | Article |
| Language: | English |
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Nature Portfolio
2017-08-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-09030-6 |
| _version_ | 1819036431821045760 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-21T08:05:25Z |
| format | Article |
| id | doaj.art-13fe765c6d834f9facf6f7bbc3879a6c |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-21T08:05:25Z |
| publishDate | 2017-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-13fe765c6d834f9facf6f7bbc3879a6c2022-12-21T19:10:48ZengNature PortfolioScientific Reports2045-23222017-08-01711810.1038/s41598-017-09030-6Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9Mark B. Nottle0Evelyn J. Salvaris1Nella Fisicaro2Stephen McIlfatrick3Ivan Vassiliev4Wayne J. Hawthorne5Philip J. O’Connell6Jamie L. Brady7Andrew M. Lew8Peter J. Cowan9Robinson Research Institute & Adelaide School of Medicine, University of AdelaideImmunology Research Centre, St. Vincent’s Hospital Melbourne, MelbourneImmunology Research Centre, St. Vincent’s Hospital Melbourne, MelbourneRobinson Research Institute & Adelaide School of Medicine, University of AdelaideRobinson Research Institute & Adelaide School of Medicine, University of AdelaideWestmead Millennium Institute, University of SydneyWestmead Millennium Institute, University of SydneyWalter and Eliza Hall Institute, MelbourneWalter and Eliza Hall Institute, MelbourneImmunology Research Centre, St. Vincent’s Hospital Melbourne, MelbourneAbstract 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.https://doi.org/10.1038/s41598-017-09030-6 |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-017-09030-6 |
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