Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: ( a ) Illustration of sgRNA-guided HIV-1 genome regulation platform for dCas9 fusion with transcriptional activator (VP64). ( b ) Diagram of dCas9-VP64 expressing lentiviral or retroviral vector and sgRNA expressing lentiviral vector (LV). ( c ) Illustration of sgRNA targeting locations within HIV-1 LTR. Some of transcription factors are shown in different colors. ( d , e ) Identification of effective sgRNAs with dCas9-VP64 in HEK293T cells transiently coexpressing NL4-3-EcoHIV- firefly luciferase (eLuc) reporter ( d ) or TZM-bI cells integrated with LTR-eLuc reporter ( e ). At 2 days after cotransfection, ONE-Glo™ luciferase assay was performed. Data represent mean ± SEM of 4 independent transfections, showing fold changes in luminescent reporter activity relative to corresponding empty sgRNA expression vector (LTR-0). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Expressing, Plasmid Preparation, Luciferase, Cotransfection, Transfection, Activity Assay

Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: ( a ) Illustration of SAM-VPH complex derived from dCas9-VP64 (V) and MS2-p65-HSF1 (PH) with msgRNA. ( b ) Similar activation of transfected EcoHIV-eLuc reporter by inducible (TRE) or constitutive (LTR) dCas9-VP64 expression in HEK293T cells. ( c ) Similar reactivation of latent HIV-LTR-eLuc reporter in TZM-bI cells after either transient plasmid transfection by Lipofectamine 3000 or stable infection by lentivirus. Note that MS2-p65-HSF1 alone did not influence the basal activity of LTR-eLuc reporter. The msgRNA-J has no effect due to 2 nucleotide mismatches to the LTR sequence from TZM-bI cells. ( d ) Identification of msgRNA LTR-L and O with best efficiency to activate EcoHIV-eLuc reporter in HEK239T cells with dCas9-SAM system. Data represent mean ± SEM of 4–6 independent transfections or infections, showing fold changes relative to corresponding empty msgRNA expression vector (LTR-0). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Derivative Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Infection, Activity Assay, Sequencing

Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: ( a,b ) TZM-bI cells were infected with dCas9-VPH and indicated msgRNA lentiviruses at 1 or 10 MOI and luciferase activity was measured at 2–21 days post-infection (dpi). ( c,d ) Indicated HIV-1 EGFP reporter T cell lines were infected with dCas9-VPH and indicated msgRNAs lentiviruses at 10 MOI and flow cytometric analysis for EGFP percentage ( c ) and individual intensity ( d ) was performed at 1 dpi. ( e,f ) HIV-1 latent CHME5 microglial cells were infected at 10 MOI and flow cytometry was performed at 1 dpi. All data represent relative changes (n = 3–4) to the corresponding empty msgRNA control (Zero). *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Infection, Luciferase, Activity Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: Potent and persistent reactivation of HIV-1 EGFP reporter virus and suicide cell death by dCas9-VPH/msgRNA in CHME5 microglial cells ( a ) and Jurkat-derived 2D10 ( b ) or E4 ( c ) T cell lines. ( a ) Time-dependent reduction in EGFP + CHME5 cells due to continuous reactivation and suicide cell death. P/H/Z triple selection enriched lentivirus-infected cells and validated continuous EGFP reporter reactivation. **p < 0.01 indicate statistical significance by student’s t test as compared to corresponding non-selection groups. ( b , c ) Time-dependent reduction in EGFP + 2D10 or E4 cells due to continuous reactivation and suicide cell death.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Derivative Assay, Selection, Infection

Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: ( a–c ) The dCas9-VPH stable TZM-bI cells were treated with SAHA or infected with indicated msgRNA lentivirus for 2–4 d before luminescence reporter assay ( a ) and CellTiter-Glo ® luminescent cell viability assay ( b ). Relative reactivation change was normalized by cell viability ( c ). ( d–g ) The dCas9-VPH stable CHME5 cells were treated with SAHA or infected with indicated msgRNA lentivirus for 4 d before EGFP flow cytometry for reactivation efficiency ( d ), total ( e ) and individual ( f ) fluorescent intensity and survival cell number ( g ). All data represent mean ± SEM of 3–4 independent experiments. *p < 0.05 and **p < 0.01 indicate statistical significance by ANOVA and FLSD test as compared with corresponding vehicle control or LTR-zero control.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Infection, Reporter Assay, Cell Viability Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

doi: 10.1038/srep16277

Figure Lengend Snippet: Overview of the dCas9-VPH SAM system using MS2-mediated sgRNA to direct multiple activators to the enhancer region of the HIV-1 promoter and reactivate viral protein expression in HIV-1 latent cells, which further induce suicide death and/or trigger host immune response to kill HIV-1 latent cells.

Article Snippet: The lentiviral vector pLV-EF1α-dCas9-VP64-BFP was generated by cloning the Bgl II/ Xho I-digested fragment of dCas9-VP64-BFP from retroviral vector pMSCV-LTR-dCas9-VP64-BFP (Addgene plasmid #46913) into lentiviral vector pLV-EF1α-Cas9-T2A-RFP via Bam HI/ Sal I (Biosettia Inc).

Techniques: Expressing