title: A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures.
abstract: Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent inhibition of virus replication was reported, HIV-1 can escape from a single antiviral gRNA by mutation of the target sequence. Here, we demonstrate that combinations of two antiviral gRNAs delay viral escape, and identify two gRNA combinations that durably block virus replication. When viral escape is prevented, repeated Cas9 cleavage leads to saturation of major mutations in the conserved target sequences that encode critical proteins. This hypermutation coincides with the loss of replication-competent virus as scored in sensitive co-cultures with unprotected cells, demonstrating complete virus inactivation. These results provide a proof-of-principle that HIV-1-infected cells can be functionally cured by dual-gRNA CRISPR/Cas9 treatment.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
authors.forename: Na
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands. Electronic address: b.berkhout@amc.uva.nl.
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands. Electronic address: a.t.das@amc.uva.nl.
authors.forename: Atze T
authors.initials: AT
authors.lastname: Das
mesh_terms: D064113:CRISPR-Cas Systems; D004279:DNA, Viral / Q000235:genetics*; D015658:HIV Infections / Q000235:genetics* / Q000628:therapy / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D009154:Mutation; D011533:Proviruses / Q000235:genetics* / Q000254:growth & development; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013601:T-Lymphocytes / Q000821:virology; D014779:Virus Replication / Q000235:genetics
chemical_list: D004279:DNA, Viral; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; Cas9; HIV-1; functional cure; preventing escape; provirus inactivation