title: Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia bDepartment of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. *Kamel Khalili, Wenhui Hu and Yonggang Zhang are senior authors and have equally contributed to this manuscript.
authors.forename: Chaoran
authors.initials: C
authors.lastname: Yin
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Raj
authors.initials: R
authors.lastname: Putatunda
authors.forename: Won-Bin
authors.initials: WB
authors.lastname: Young
authors.forename: Kamel
authors.initials: K
authors.lastname: Khalili
authors.forename: Wenhui
authors.initials: W
authors.lastname: Hu
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy / Q000821:virology*; D019380:Anti-HIV Agents / Q000378:metabolism*; D001688:Biological Products / Q000378:metabolism*; D019295:Computational Biology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005820:Genetic Testing; D016679:Genome, Viral; D060005:Genotyping Techniques; D057809:HEK293 Cells; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000187:drug effects / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017735:Virus Latency / Q000187:drug effects
chemical_list: D019380:Anti-HIV Agents; D001688:Biological Products; D000094704:RNA, Guide, CRISPR-Cas Systems; D008156:Luciferases