Specific Reactivation of Latent HIV-1 by dCas9-SunTag-VP64-mediated Guide RNA Targeting the HIV-1 Promoter

Abstract

HIV-1 escapes antiretroviral agents by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. Transcriptional activation is prerequisite for reactivation and the eradication of latent HIV-1 proviruses. dCas9-SunTag-VP64 transcriptional system has been reported that it can robustly activate the expression of an endogenous gene using a single guide RNA (sgRNA). Here, we systematically investigated the potential of dCas9-SunTag-VP64 with the designed sgRNAs for reactivating latent HIV-1. We found dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 targeted from -164 to -146 or -124 to -106 bp upstream of the transcription start sites of HIV-1 could induce high expression of luciferase reporter gene after screening of sgRNAs targeting different regions of the HIV-1 promoter. Further, we confirmed that dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 can effectively reactivate latent HIV-1 transcription in several latently infected human T-cell lines. Moreover, we confirmed that the reactivation of latent HIV-1 by dCas9-SunTag-VP64 with the designed sgRNA occurred through specific binding to the HIV-1 LTR promoter without genotoxicity and global T-cell activation. Taken together, our data demonstrated dCas9-SunTag-VP64 system can effectively and specifically reactivate latent HIV-1 transcription, suggesting that this strategy could offer a novel approach to anti-HIV-1 latency.

Specific and Stable Suppression of HIV Provirus Expression In Vitro by Chimeric Zinc Finger DNA Methyltransferase 1

“…YA cells and C11 cells, which are HIV-1-infected or latently infected Jurkat T cells, respectively, were first created in our laboratory and are now used widely in the research community 36, 37, 51, 52, 53, 54. The C11 and YA cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U mL −1 penicillin, and 100 μg mL −1 streptomycin (Invitrogen) at 37°C under 5% CO 2 .…”

Genome-Editing Technologies: Principles and Applications

“…dCas9 transcriptional effectors have even been used to efficiently mediate repression and activation of endogenous genes in Drosophila (Lin et al 2015b) and in plant cells (Piatek et al 2015). Both TALE and Cas9 activators have also been configured to stimulate transcription of latent HIV Ji et al 2016;Limsirichai et al 2016;Perdigao et al 2016;Saayman et al 2016), indicating their potential to work in concert with antiretroviral therapy for eradicating HIV infection. Importantly, because of the ease with which the CRISPRCas9 system can be used, genome-wide screens using Cas9 transcriptional activators (Gilbert et al 2014;Konermann et al 2015) and repressors (Gilbert et al 2014) can be easily implemented to discover genes involved in a number of diverse processes, including drug resistance and cancer metastasis.…”

Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome

“…They showed that targeting of different regions of LTR particularly LTR promoter could lead to activation of viral gene expression in cell line models of HIV-1 latency. [123][124][125][126] 2 | DISCUSSION Genome editing using CRISPR/Cas9 represents a powerful strategy to treat various diseases including viral infections. In this study, we provide key insights into the mechanism and application of CRISPR/ Cas9 in eradication and limitation of several DNA viruses from different cell lines.…”

“…Several studies employed CRISPR/dCas9 system (a deactivated Cas9 nuclease bonded to transcription activators) to induce activation of dormant HIV provirus. They showed that targeting of different regions of LTR particularly LTR promoter could lead to activation of viral gene expression in cell line models of HIV‐1 latency …”