A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9
The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2015-01-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S232905011630047X |
| _version_ | 1819036668781395968 |
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| author | Ming Yuan Xuefei Gao Louisa S Chard Zarah Ali Jahangir Ahmed Yunqing Li Pentao Liu Nick R Lemoine Yaohe Wang |
| author_facet | Ming Yuan Xuefei Gao Louisa S Chard Zarah Ali Jahangir Ahmed Yunqing Li Pentao Liu Nick R Lemoine Yaohe Wang |
| author_sort | Ming Yuan |
| collection | DOAJ |
| description | The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (â¼90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application. |
| first_indexed | 2024-12-21T08:09:11Z |
| format | Article |
| id | doaj.art-877a270d03ca43068bd8606fc497f5da |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-21T08:09:11Z |
| publishDate | 2015-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-877a270d03ca43068bd8606fc497f5da2022-12-21T19:10:43ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012015-01-012A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9Ming Yuan0Xuefei Gao1Louisa S Chard2Zarah Ali3Jahangir Ahmed4Yunqing Li5Pentao Liu6Nick R Lemoine7Yaohe Wang8Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UKWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UKCentre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UKThe Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, London, UKCentre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UKCentre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UKWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UKCentre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK; Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University, Zhengzhou, ChinaCentre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK; Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University, Zhengzhou, ChinaThe current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (â¼90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.http://www.sciencedirect.com/science/article/pii/S232905011630047X |
| spellingShingle | Ming Yuan Xuefei Gao Louisa S Chard Zarah Ali Jahangir Ahmed Yunqing Li Pentao Liu Nick R Lemoine Yaohe Wang A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 Molecular Therapy: Methods & Clinical Development |
| title | A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 |
| title_full | A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 |
| title_fullStr | A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 |
| title_full_unstemmed | A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 |
| title_short | A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9 |
| title_sort | marker free system for highly efficient construction of vaccinia virus vectors using crispr cas9 |
| url | http://www.sciencedirect.com/science/article/pii/S232905011630047X |
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