We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.