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Objective: There is an urgent need for the development of HIV-1 genome eradication strategies that lead to a permanent cure for HIV-1/AIDS. We previously reported that four guide RNAs (gRNAs) targeting HIV-1 long terminal repeats (LTR) effectively eradicated the entire HIV-1 genome. In this study, we sought to identify the best gRNAs targeting HIV-1 LTR and viral structural region and optimize gRNA pairing that can efficiently eradicate the HIV-1 genome., Design: Highly specific gRNAs were designed using bioinformatics tools, and their capacity of guiding CRISPR-associated system 9 to cleave HIV-1 proviral DNA was evaluated using high-throughput HIV-1 luciferase reporter assay and rapid Direct-PCR genotyping., Methods: The target seed sequences for each gRNA were cloned into lentiviral vectors. HEK293T cells were cotransfected with a pEcoHIV-NL4-3-firefly-luciferase reporter vector (1 : 20) over lentiviral vectors carrying CRISPR-associated system 9 and single gRNA or various combinations of gRNAs. The EcoHIV DNA cleaving efficiency was evaluated by Direct-PCR genotyping, and the EcoHIV transcription/replication activity was examined by a luciferase reporter assay., Results: Most of the designed gRNAs are effective to eliminate the predicted HIV-1 genome sequence between the selected two target sites. This is evidenced by the presence of PCR genotypic deletion/insertion and the decrease of luciferase reporter activity. In particular, a combination of viral structural gRNAs with LTR gRNAs provided a higher efficiency of genome eradication and an easier approach for PCR genotyping., Conclusion: Our screening strategy can specifically and effectively identify gRNAs targeting HIV-1 LTR and structural region to excise proviral HIV-1 from the host genome.