title: A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9.
abstract: 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 (~90%) 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.
authors.affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London , London, UK.
authors.forename: Ming
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus , Cambridge, UK.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Gao
authors.affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London , London, UK.
authors.forename: Louisa S
authors.initials: LS
authors.lastname: Chard
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London , London, UK.
authors.forename: Zarah
authors.initials: Z
authors.lastname: Ali
authors.affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London , London, UK.
authors.forename: Jahangir
authors.initials: J
authors.lastname: Ahmed
authors.affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London , London, UK.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Li
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus , Cambridge, UK.
authors.forename: Pentao
authors.initials: P
authors.lastname: Liu
authors.affiliation: Centre 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, China.
authors.forename: Nick R
authors.initials: NR
authors.lastname: Lemoine
authors.affiliation: Centre 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, China.
authors.forename: Yaohe
authors.initials: Y
authors.lastname: Wang