title: CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.
abstract: CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.
authors.affiliation: The Unit of Anti-Viral Immunity and Genetic Therapy, the Key Laboratory of Molecular Virology and Immunology, the Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China 200025.
authors.forename: Weiming
authors.initials: W
authors.lastname: Wang
authors.affiliation: The Unit of Anti-Viral Immunity and Genetic Therapy, the Key Laboratory of Molecular Virology and Immunology, the Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China 200025.
authors.forename: Chaobaihui
authors.initials: C
authors.lastname: Ye
authors.affiliation: The Unit of Anti-Viral Immunity and Genetic Therapy, the Key Laboratory of Molecular Virology and Immunology, the Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China 200025.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Kimata
authors.affiliation: The Unit of Anti-Viral Immunity and Genetic Therapy, the Key Laboratory of Molecular Virology and Immunology, the Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China 200025.
authors.forename: Paul
authors.initials: P
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000627:therapeutic use; D002460:Cell Line; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000627:therapeutic use; D015658:HIV Infections / Q000235:genetics / Q000628:therapy*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000627:therapeutic use; D019713:Receptors, CCR5 / Q000235:genetics*; D014161:Transduction, Genetic; D053586:Virus Internalization
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D019713:Receptors, CCR5