title: A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors.
abstract: The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.
authors.affiliation: Center for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London; m.yuan@qmul.ac.uk.
authors.forename: Ming
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University; School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University.
authors.forename: Pengju
authors.initials: P
authors.lastname: Wang
authors.affiliation: Center for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London.
authors.forename: Louisa S
authors.initials: LS
authors.lastname: Chard
authors.affiliation: Center for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London; Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University; School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Lemoine
authors.affiliation: Center for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London; Sino-British Research Centre for Molecular Oncology, National Center for International Research in Cell and Gene Therapy, Zhengzhou University; School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University; Yaohe.wang@qmul.ac.uk.
authors.forename: Yaohe
authors.initials: Y
authors.lastname: Wang
mesh_terms: D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053903:DNA Breaks, Double-Stranded; D000094704:RNA, Guide, CRISPR-Cas Systems; D014616:Vaccinia virus
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems