A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

作者：Yuan, Ming^*; Wang, Pengju; Chard, Louisa S.; Lemoine, Nicholas R.; Wang, Yaohe^*

来源：Jove-Journal of Visualized Experiments, 2016, (116): e54171.

DOI：10.3791/54171

• Infection
• Issue 116
• Vaccinia virus
• genome
• editing
• homologous recombination
• CRISPR
• Cas9

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.

• 出版日期2016-10
• 单位郑州大学