title: RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection.
abstract: AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5' to 3' LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140; kamel.khalili@temple.edu wenhui.hu@temple.edu.
authors: Wenhui
authors: W
authors: Hu
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
authors: Rafal
authors: R
authors: Kaminski
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
authors: Fan
authors: F
authors: Yang
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
authors: Yonggang
authors: Y
authors: Zhang
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
authors: Laura
authors: L
authors: Cosentino
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
authors: Fang
authors: F
authors: Li
authors: Cancer Genome Institute, Fox Chase Cancer Center, Temple University School of Medicine, Philadelphia, PA 19111;
authors: Biao
authors: B
authors: Luo
authors: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and.
authors: David
authors: D
authors: Alvarez-Carbonell
authors: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and.
authors: Yoelvis
authors: Y
authors: Garcia-Mesa
authors: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and.
authors: Jonathan
authors: J
authors: Karn
authors: Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
authors: Xianming
authors: X
authors: Mo
authors: Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140; kamel.khalili@temple.edu wenhui.hu@temple.edu.
authors: Kamel
authors: K
authors: Khalili
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D057809:HEK293 Cells; D015658:HIV Infections / Q000276:immunology / Q000517:prevention & control* / Q000821:virology*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics*; D014611:Vaccination; D017735:Virus Latency / Q000235:genetics*; D058727:RNA, Small Untranslated
chemical_list: D012313:RNA; D058727:RNA, Small Untranslated
keywords: CRISPR/Cas9; genome editing; latency; reservoir; retrovirus