title: Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells.
abstract: Genetically modified HSV-1 viruses serve as promising vectors for tumour therapy and vaccine development. The CRISPR/Cas9 system is one of the most powerful tools for precise gene editing of the genomes of organisms. However, whether the CRISPR/Cas9 system can precisely and efficiently make gene replacements in the genome of HSV-1 remains essentially unknown. Here, we reported CRISPR/Cas9-mediated editing of the HSV-1 genome in human cells, including the knockout and replacement of large genes. In established cells stably expressing CRISPR/Cas9, gRNA in coordination with Cas9 could direct a precise cleavage within a pre-defined target region, and foreign genes were successfully used to replace the target gene seamlessly by HDR-mediated gene replacement. Introducing the NHEJ inhibitor SCR7 to the CRISPR/Cas9 system greatly facilitated HDR-mediated gene replacement in the HSV-1 genome. We provided the first genetic evidence that two copies of the ICP0 gene in different locations on the same HSV-1 genome could be simultaneously modified with high efficiency and with no off-target modifications. We also developed a revolutionized isolation platform for desired recombinant viruses using single-cell sorting. Together, our work provides a significantly improved method for targeted editing of DNA viruses, which will facilitate the development of anti-cancer oncolytic viruses and vaccines.
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Chaolong
authors.initials: C
authors.lastname: Lin
authors.affiliation: School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Li
authors.affiliation: School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Mengru
authors.initials: M
authors.lastname: Hao
authors.affiliation: School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Chenghao
authors.initials: C
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, 361102, China.|School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems