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Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a “shock and kill” strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.

Objective There is an urgent need for the development of HIV-1 genome eradication strategies that lead to a permanent cure for HIV-1/AIDS. We previously reported that 4 guide RNAs (gRNAs) targeting HIV-1 long terminal repeats (LTR) effectively eradicated the entire HIV-1 genome. In this study, we sought to identify the best gRNAs targeting HIV-1 LTR and viral structural region and to optimize gRNA pairing that can efficiently eradicate the HIV-1 genome. Design Highly specific gRNAs were designed using bioinformatics tools and their capacity of guiding Cas9 to cleave HIV-1 proviral DNA was evaluated using high throughput HIV-1 luciferase reporter assay and rapid Direct-PCR genotyping. Methods The target seed sequences for each gRNA were cloned into lentiviral vectors. HEK293T cells were cotransfected with a pEcoHIV-NL4-3-firefly-luciferase reporter vector (1:20) over lentiviral vectors carrying Cas9 and single gRNA or various combinations of gRNAs. The EcoHIV DNA cleaving efficiency was evaluated by Direct-PCR genotyping and the EcoHIV transcription/replication activity was examined by a luciferase reporter assay. Results Most of the designed gRNAs are effective to eliminate the predicted HIV-1 genome sequence between the selected two target sites. This is evidenced by the presence of PCR genotypic deletion/insertion and the decrease of luciferase reporter activity. In particular, a combination of viral structural gRNAs with LTR gRNAs provided a higher efficiency of genome eradication and an easier approach for PCR genotyping. Conclusion Our screening strategy can specifically and effectively identify gRNAs targeting HIV-1 LTR and structural region to excise proviral HIV-1 from the host genome.