title: Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.
abstract: CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors: Chang
authors: C
authors: Li
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors: Xinmeng
authors: X
authors: Guan
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors: Tao
authors: T
authors: Du
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors: Wei
authors: W
authors: Jin
authors: Department of General Surgery, Wuhan No.1 Hospital, Wuhan 430022, PR China.
authors: Biao
authors: B
authors: Wu
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors: Yalan
authors: Y
authors: Liu
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors: Ping
authors: P
authors: Wang
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors: Bodan
authors: B
authors: Hu
authors: Institute for Infection and Immunity, St George's University of London, London SW17 0RE, UK.
authors: George E
authors: GE
authors: Griffin
authors: Section of Infectious Diseases, Faculty of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors: Robin J
authors: RJ
authors: Shattock
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|Institute for Infection and Immunity, St George's University of London, London SW17 0RE, UK.
authors: Qinxue
authors: Q
authors: Hu
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D019713:Receptors, CCR5 / Q000235:genetics* / Q000378:metabolism
chemical_list: C584933:CCR5 protein, human; D019713:Receptors, CCR5