Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS. (15th May 2016)

• Record Type: Journal Article
• Authors: Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Fan; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui; Zhang, Yonggang
• Is Part Of: AIDS. Volume 30:Number 8(2016)
• Page Start: 1163
• Page End: 1174
• Publication Date: 2016-05-15
• Subjects: CRISPR/Cas9 -- cure -- eradication -- gRNA -- HIV -- latent reservoir; AIDS (Disease) -- Periodicals; Acquired Immunodeficiency Syndrome; AIDS (Disease); Periodicals; Periodicals; 616.9792005
• DOI: 10.1097/QAD.0000000000001079
• Languages: English
• ISSNs: 0269-9370

Abstract

Abstract : Supplemental Digital Content is available in the text Abstract : 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.