Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation
The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem...
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MDPI AG
2020-06-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/11/6/713 |
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| author | Natalia Ryczek Magdalena Hryhorowicz Daniel Lipiński Joanna Zeyland Ryszard Słomski |
| author_facet | Natalia Ryczek Magdalena Hryhorowicz Daniel Lipiński Joanna Zeyland Ryszard Słomski |
| author_sort | Natalia Ryczek |
| collection | DOAJ |
| description | The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (<i>Sus scrofa domestica</i>) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine <i>GGTA1</i>, <i>CMAH</i>, <i>β4GalNT2</i>, <i>vWF</i>, <i>ASGR1</i> genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies. |
| first_indexed | 2024-03-10T18:51:02Z |
| format | Article |
| id | doaj.art-207d8aea025a447dae16cf32983acd04 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-10T18:51:02Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-207d8aea025a447dae16cf32983acd042023-11-20T05:04:58ZengMDPI AGGenes2073-44252020-06-0111671310.3390/genes11060713Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation FormationNatalia Ryczek0Magdalena Hryhorowicz1Daniel Lipiński2Joanna Zeyland3Ryszard Słomski4Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznań, PolandDepartment of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznań, PolandDepartment of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznań, PolandDepartment of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznań, PolandInstitute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, PolandThe increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (<i>Sus scrofa domestica</i>) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine <i>GGTA1</i>, <i>CMAH</i>, <i>β4GalNT2</i>, <i>vWF</i>, <i>ASGR1</i> genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.https://www.mdpi.com/2073-4425/11/6/713xenoantigencoagulation system dysregulationCRISPR/Cas9 systemgenome modificationsnon-homologous DNA ends joining (NHEJ)TIDE analysis |
| spellingShingle | Natalia Ryczek Magdalena Hryhorowicz Daniel Lipiński Joanna Zeyland Ryszard Słomski Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation Genes xenoantigen coagulation system dysregulation CRISPR/Cas9 system genome modifications non-homologous DNA ends joining (NHEJ) TIDE analysis |
| title | Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation |
| title_full | Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation |
| title_fullStr | Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation |
| title_full_unstemmed | Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation |
| title_short | Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation |
| title_sort | evaluation of the crispr cas9 genetic constructs in efficient disruption of porcine genes for xenotransplantation purposes along with an assessment of the off target mutation formation |
| topic | xenoantigen coagulation system dysregulation CRISPR/Cas9 system genome modifications non-homologous DNA ends joining (NHEJ) TIDE analysis |
| url | https://www.mdpi.com/2073-4425/11/6/713 |
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