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. 2016 Feb 16;24(3):508–521. doi: 10.1038/mt.2016.7

Figure 7.

Figure 7

dCas9-SunTag-VP64 specifically binds to the LTR to reactivate latent HIV-1. (a) Schematic representation of the deletion of the sgRNA 4 target site in the LTR-luc reporter vector aligned with wild type of LTR-luc vector. (b) Cotransfection of dCas9-SunTag-VP64 with sgRNA 4 or with sgRNA negative vector and the LTR-luc or LTR(ΔsgRNA4)-luc reporter plasmids into HEK293T cells at the indicated time. The luciferase activity induced by dCas9-SunTag-VP64 with sgRNA 4 was normalized to that of sgRNA-negative vector in each group at 72 hours post-transfection. The data represent the mean ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001; paired t-test. (c) Analysis of dCas9-SunTag-VP64 with sgRNA 4-specific binding to the HIV-1 5′-LTR promoter by a chromatin immunoprecipitation assay. C11 cells were nucleofected with dCas9-SunTag-VP64 and indicated sgRNA at the indicated time. Chromatin fragments were immunoprecipitated with anti-HA antibody or normal mouse IgG and then amplified by primers specific for HIV-1 5′-LTR promoter. (d) Analysis of the fold change of immunoprecipitated chromatin fragments from cells treated with dCas9-SunTag-VP64 with sgRNA 4 or with sgRNA negative vector group by quantitative polymerase chain reaction. The signal was normalized to that of input in each group. The data represent the mean ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001; paired t-test.