Of the 12 million people who inject drugs worldwide, 13% are chronically infected with Human Immunodeficiency Virus (HIV), i.e., they live with HIV. Chronic opioid use affects the host immune system and increases an individual’s susceptibility to HIV infection. However, it is unclear how opioid use changes the course of HIV pathogenesis. Particularly, there is a gap in understanding how opioids impact HIV latency. Latency results in a reservoir of infected quiescent cells that evade antiviral immune responses, are not targeted by antiretroviral therapy (ART), and allow HIV viremia to rebound upon treatment interruption. While in vitro studies show that opioids modulate the activity of transcription factors involved in T-cell activation and HIV transcription, few studies have investigated whether opioid use impacts HIV latency in vivo in HIV-infected people. In this research, peripheral blood mononuclear cells (PBMCs) were utilized from People with HIV (PWH) with or without recent opioid use or opioid use disorder (OUD) who were enrolled in the Linking Infection and Narcology Care-Part II (LINC-II) and Studying Partial Agonists for Ethanol and Tobacco Elimination in Russians with HIV (St PETER HIV ARCH) studies conducted in St. Petersburg, Russia. Intact proviral DNA digital droplet PCR (ddPCR) assays were performed on PBMCs from antiretroviral treated PWH, with (n=8) or without (n=11) current OUD, to quantify intact and defective proviral genomes. Samples from ART-treated PWH with OUD compared to those without OUD had similar levels of intact and defective proviruses. To evaluate latency reversal, PBMCs from ART-treated PWH with or without OUD, were activated with anti-CD3/28 beads and RT-ddPCR assays were performed to measure HIV LTR-gag RNA. A variable response in PWH without OUD was seen where half of the samples showed an increase in HIV RNA upon activation. Interestingly, only 1 of 8 samples from PWH with OUD showed an increase in HIV transcription. However, no suppression of HIV reactivation was found in vitro from latent cells generated using a primary CD4+ T-cell latency model in the presence or absence of morphine. Similarly, no differences in HIV integration and transcription in vitro were observed between morphine and control conditions. Additionally, expression of opioid receptors was not detected in primary PBMCs, CD4 T cells, or macrophages. These results show that PWH with OUD have a pool of persistent HIV proviruses that are refractive to reactivation, although opioids did not affect HIV replication and latency reactivation in vitro. The discrepancy in these in vitro and in vivo results and the lack of expression of opioid receptors in immune cells suggests that while opioids do not directly impact HIV replication, latency, and reactivation in target CD4+ cells, opioids could indirectly shape the HIV reservoir in vivo by modulating general immune functions, neuroderived factors or other cells that are responsive to opioids. Eradication of the latent HIV reservoir is necessary to achieve a cure for HIV/AIDS. One approach for latency eradication is the “shock and kill” approach that entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. An alternative approach is to permanently repress HIV expression precluding viral rebound after ART discontinuation. Here, a nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB) was used to epigenetically silence the proviral DNA. I show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, which are histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies. The research presented here provides evidence of opioid-mediated modulation of HIV-1 latency reactivation in PWH with opioid dependency. Additionally, we show that HIV-1 reactivation can be suppressed by epigenetic remodeling of the HIV-1 promoter using a repurposed CRISPR/Cas9 system.