The entry of HIV-1 into the brain during the early and/or late stages of infection leads to the development of a viral reservoir in the vast majority f individuals despite effective combination antiretroviral therapy (cART). In this regard, HIV-1 proviral DNA integration into the chromosomes of brain perivascular macrophages, microglial cells, astrocytes, and perhaps brain endothelial cells leads to the establishment of viral latency/persistence within these important cellular compartments as well as resting memory T cells and cells of the monocyte-macrophage lineage in the peripheral circulation and likely other tissues during the course of HIV disease. The extent of viral gene activation and expression in the periphery and following viral brain penetration may be dependent on therapeutic efficacy in a given reservoir, the diversity of the viral quasispecies, host immune activation profiles, and a number of comorbidity factors such as substance abuse, aging, and other chronic infections or cancers. To varying degrees, these factors are integrally involved in modulating the production of full-length and truncated viral RNA, toxic viral proteins (Tat, Nef, Vpr, and gp120), and infectious virus within and outside the brain. Consequently, there is a critical need for new strategies to eliminate all forms of integrated provirus from latently/persistently infected cells thereby preventing infectious production as well as the production of neurotoxic viral proteins that could also be produced from defective genomes not eliminated by currently available therapeutic strategies. To this end, a team experienced investigators has been assembled with complementary expertise in viral diversity and molecular architecture of the HIV-1 genome (B. Wigdahl, Drexel University), viral latency (J. Karn, Case Western Reserve University), and gene excision technology (K. Khalili, Temple University) to examine the Hypothesis that the CRISPR/Cas9 gene editing platform can be tailored to develop precision-guided gene editing strategies to eliminate HIV-1 from the latently infected resting memory CD4+ T-cell reservoir and reservoir cells with the brain. To address this hypothesis, three specific aims are proposed. In Aim 1 sequence and bioinformatic information from viral genetic studies performed with HIV-1-infected patients will be utilized to design gRNAs to precisely guide the HIV-1 excision process (Drexel University). In Aim 2 we will develop and test in vitro HIV-1- specific gene editing systems (Temple University), combined with expertise in the molecular biology of HIV-1 latency (Case Western Reserve University), which will culminate in ex vivo experimentation in Aim 3 on HIV-1- infected samples. These studies will set the stage for future in vivo animal studies for validating the approach toward clinical application. The overarching goal is to develop a robust experimental procedure which can be employed through various gene delivery platforms, such as nanomolecules, lentivirus, or stem/progenitor cell engineering, to provide a cure for AIDS.