title: CRISPR-Cas9 system-driven site-specific selection pressure on Herpes simplex virus genomes.
abstract: The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China; Centre for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: liwisch@gmail.com.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Li
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China; Centre for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: byw1004@126.com.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Bi
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China; Centre for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: xiao.hongjian@imbcams.com.cn.
authors.forename: Hongjian
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Kunming Medical University, 1168 West Chunrong Road, Kunming, Yunnan, 650500, China. Electronic address: pisceseva347@outlook.com.
authors.forename: Le
authors.initials: L
authors.lastname: Sun
authors.affiliation: Baoshan College of Traditional Chinese Medicine, No. 20, The sixth Community, Yaowan Village, Longyang District, Baoshan, Yunnan, 678000, China. Electronic address: 81024703@qq.com.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ren
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China. Electronic address: yadong_li@imbcams.com.cn.
authors.forename: Yadong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China. Electronic address: chenchen9010@gmail.com.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, 935 Jiaoling Road, Wuhua District, Kunming, Yunnan, 650118 China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China; Centre for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: cunwei@foxmail.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Cun
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002522:Chlorocebus aethiops; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D017930:Genes, Reporter; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic; D014709:Vero Cells; D014779:Virus Replication
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I
keywords: CRISPR-Cas9; HSV-1; Selection pressure; Site-specific mutation; Viral genome replication