CRISPR-Cas13a Inhibits HIV-1 Infection

Figures

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  Figure 1. Cas13a/crRNA Inhibits HIV-1 Gene Expression and Virus Production from the Transfected Plasmid DNA (A) Illustration of the crRNAs that target HIV-1 genome. Positions of the nucleotide refer to the HIV-1 (NL4-3) genome. Arrows indicate location of the crRNA. (B) Western blotting to measure the expression of LbuCas13a. (C) Cas13a/crRNA diminishes HIV-1 gene expression. HEK293T cells were co-transfected with LbuCas13a/crRNA and pNL4-3-ΔE-YFP plasmid DNA. 48 h post transfection, cells were harvested and YFP expression from HIV-1 LTR promoter was monitored by flow cytometry. (D) MFI (mean fluorescence intensity) of YFP. The MFI value in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (E) MFI of YFP. The MFI value in cells that were treated with LTR1 was arbitrarily set as 1. (F) Effect of Cas13a/crRNA on HIV-1 Gag expression, measured by western blotting. (G) HIV mRNA was quantified by real-time RT-PCR. Results obtained with the empty crRNA vector were arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (H) Cas13a/crRNA inhibits virus production.…

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  Figure 2. Cas13a/crRNA Inhibits HIV-1 Infection (A and B) HEK293T cells were transfected with the Cas13a/crRNA plasmid DNA. 6 h after transfection, cells were infected with HIV-1 (carrying YFP reporter) for 36 h. YFP expression from the infected HIV-1 was measured by flow cytometry. Representative histograms are shown in (A). Values of YFP MFI are shown in (B). YFP MFI in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (C) HIV-1 RNA was quantified by real-time RT-PCR. Results obtained with the empty crRNA vector were arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (D) YFP MFI in cells that were treated with LTR1 crRNA was arbitrarily set as 1. Error bars indicate SD. (E) Cas13a/crRNA impairs virus production. Viruses in the supernatants were harvested by ultracentrifugation, followed by western blotting to measure levels of HIV-1 Gag protein. (F) Cas13a/crRNA attacks the HIV-1 RNA entering into cells. HEK293T cells were transfected with Cas13a/crRNA and infected with HIV-1. 3 h after infection, real-time RT-PCR was performed to measure levels of HIV-1…

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  Figure 3. Cas13a/crRNA Inhibits HIV-1 Infection in Jurkat Cells (A and B) Jurkat cells were transfected with the Cas13a/crRNA plasmid DNA. 6 h after transfection, cells were infected with HIV-1 for 72 h. p24 expression from the infected HIV-1 was measured by flow cytometry. Cells were first gated by FSC/SSC to exclude debris, FLAG+/p24+ cells were gated for p24 expression analysis. Representative histograms are shown in (A). Values of p24 MFI are shown in (B). p24 MFI in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test).

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  Figure 4. Cas13a/crRNA Impairs EGFP Expression from DNA that Is Integrated into Cellular DNA (A and B) Cas13a/crRNA was transfected into HEK293T cells that bear the stably integrated LTR-EGFP. EGFP expression was measured by flow cytometry. Representative histograms are shown in (A). Values of YFP MFI are shown in (B). YFP MFI in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (C) EGFP mRNA was quantified by real-time RT-PCR. Results obtained with the empty crRNA vector were arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test)

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  Figure 5. Cas13a/crRNA Inhibits HIV-1 Gene Expression and Virus Production from Latent HIV-1 (A) JLat10.6 cells were transfected with Cas13a/crRNA. EGFP expression was measured by flow cytometry. Results obtained with the empty crRNA vector were arbitrarily set as 1. YFP MFI in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (B) Levels of HIV-1 RNA were measured using TaqMan PCR in JLat10.6 cells transfected with Cas13a/crRNA. YFP MFI in cells that were treated with empty crRNA was arbitrarily set as 1. Error bars indicate SD (n = 3; ∗p < 0.05; ∗∗p < 0.01; Dunnett’s t test). (C) Levels of viruses in the supernatants from JLat10.6 cells were determined by western blotting with anti-HIV-1 p24/Gag protein.