title: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex.
abstract: HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.|HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Sheena M
authors.initials: SM
authors.lastname: Saayman
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Lazar
authors.affiliation: HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Tristan A
authors.initials: TA
authors.lastname: Scott
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Hart
authors.affiliation: Division of Molecular Biology, Beckman Research Institute at the City of Hope, Duarte, California, USA.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Division of Molecular Biology, Beckman Research Institute at the City of Hope, Duarte, California, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Burnett
authors.affiliation: Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA.
authors.forename: Vicente
authors.initials: V
authors.lastname: Planelles
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.|School of Biotechnology and Biomedical Sciences, University of New South Wales, Kensington, New South Wales, Australia.
authors.forename: Kevin V
authors.initials: KV
authors.lastname: Morris
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.|HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.|Wits/SA MRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D015658:HIV Infections / Q000378:metabolism / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D059372:Nucleotide Motifs; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015533:Transcriptional Activation; D014775:Virus Activation; D017735:Virus Latency
chemical_list: D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D016328:NF-kappa B; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases