Detailed protocols for materials and methods are provided in Supplementary Information.
Cloning and transfection
CRISPR plasmid constructs containing mosaic gRNAs were cloned by T4 ligase oligonucleotide insertion (Table S5) in px333 (Addgene #64073) or pLentiCRISPR-RFP657 (Addgene #75162) and transformed into STBL3 E. coli. In Fusion HD Cloning (Takara #638920) was utilized to synthesize tat mutants on pHIV-1NL4-3-Δnef-eGFP background. Maxiprep purified plasmids were transfected into HEK293FT cells using PEI or into ACH2 / U1 leukocytes via TransIT-Jurkat reagent (Mirus #2120). Supernatants were collected 72 hours after media replacement (PEI) or plasmid transfection (TransIT-Jurkat).
Virus, transduction, and viral outgrowth assays
HIV-1NL4-3-Δnef-eGFP and HIV-1NL4-3-Δnef-eGFP-Δtat were synthesized by reverse-genetics from supernatants of transfected HEK293FT cells. Supernatants were passed through 0.45 μm filters and ultracentrifuged to purify and concentrate virus. HIV-1 viral stocks were titered by RT-qPCR (Takara #631235) to determine viral RNA copies / mL. Multiplicities of infection (MOI) for HIV-1 infection of CEM-SS T cells (NIH ARP #776) were calculated as viral RNA copies / cell. CRISPR-transducing lentivirus was commercially prepared at the University of Iowa Viral Vector Core Facility and titered using digital droplet PCR. ACH2 T cells (NIH ARP #349) were transduced with CRISPR-encoding lentivirus at designated MOIs such that MOI 1 = 1 transducing unit per cell. ACH2 and U1 (NIH ARP #165) were stimulated 3 days post transfection or transduction with recombinant tumor necrosis factor alpha (TNFα; 15 ng / mL) or phorbol 12-myristate 13-acetate (PMA; 20 ng / mL) for 3 additional days followed by experimental harvest.
DNA was harvested from experimental pooled biological triplicates using NucleoSpin Tissue XS Micro kit (Machery-Nagel #740901). PCR for proviral DNA excision was run in 25 μL reactions using PrimeTime Gene Expression Master Mix (IDT #1055772) using 150 ng template DNA and 35 cycles or 15 cycles plus 35 cycles for nested PCR. Off-target PCRs were performed with 150 ng template DNA in 25 μL reactions using AmpliTaq Gold 360 master mix. Primer sequences for all reactions are provided in Table S6. Gel extracted PCR amplicons and total PCR contents were sequenced and subjected to Synthego Inference of CRISPR Edits (ICE) Analysis v2.0 (https://ice.synthego.com/#/).
RNA was harvested from pooled biological triplicates using TRIzol™ solution (Invitrogen #15596026), DNase I digested (Zymo Research #E1010), and converted to cDNA (Thermo Scientific #AB1453B). RT-qPCR was performed in technical triplicates using standard curve absolute quantitation for gapdh (1 diploid genome = 6.6 picograms DNA extracted from ACH2) and spCas9 (pLenti-CRISPR-RFP657; 109 copies equal 12.01 ng according to MW 7.22 × 106 Da). Reactions were performed in 10 μL volumes containing 1 μL cDNA templated, 5 μL PrimeTime Gene Expression Master Mix, 500 nM of each primer (5′-CCCAAGAGGAACAGCGATAAG-3’; 5′-CCACCACCAGCACAGAATAG-3’), and 0.5 μL 20x GAPDH endogenous control probe (Applied Biosystems #4333764T) or 200 nM spCas9 probe (56-FAM/ATCGCCAGA/ZEN-/AAGAAGGACTGGGAC/3IABkFQ).
Reverse transcriptase activity assay
Culture supernatants were assayed for RT activity by surveying for 3H-thymidine incorporation (3H-TTP; Perkin Elmer #NET221A001MC). Briefly, 10 μL of culture supernatants were spiked into 96-well round bottom plates, digested for 15 minutes with 10 μL solution A (100 mM Tris-HCl pH 7.9, 300 mM KCl, 10 mM DTT, 0.1% NP-40), then reacted with 25 μL solution B (50 mM Tris-HCl pH 7.9, 150 mM KCl, 5 mM DTT, 15 mM MgCl2, 0.05% NP-40, 0.250 U/mL oligo dt pd(T)12-18), 10 μCi / mL 3H-TTP (4 μL / mL; 3H-deoxythimidine 5′-triphosphate, tetrasodium salt, (methyl-3H)). After 20 hours incubations, 50 μL ice cold 10% tricholoroacetic acid was added to plates, vacuum filtered across 96-well MicroHarvest Plates, and red by scintillation counting using TopCount Scintillation counter (Perkin Elmer).
Experiments were un with biological replicates (n = 3 or 4) in at least two separate trials. Data represent mean ± standard error of the mean (SEM). Pearson correlation, one-way ANOVA with Dunnet correction, and two-way ANOVA with Sidak correction for multiple comparisons were calculated using GraphPad Prism v7.0a for Mac OS X (GraphPad Software, San Diego, California USA, www.graphpad.com).
Article TitleExonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination
A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high mutation rate of HIV-1 gives rise to numerous circulating strains with increased capacity for immune evasion and antiretroviral drug resistance. To facilitate viral elimination while accounting for this diversity, we propose genetic inactivation of proviral DNA with CRISPR-spCas9. We designed a library of “mosaic gRNAs” against a HIV-1 consensus sequence constructed from 4004 clinical strains, targeting the viral transcriptional regulator tat. Testing in 7 HIV-1 transmitted founder strains led, on average, to viral reductions of 82% with tandem TatD and TatE (TatDE) treatment. No off-target cleavages were recorded. Lentiviral transduction of TatDE attenuated latency reversal by 94% in HIV-infected, transcriptionally silent ACH2 T cells. In all, TatDE guide RNAs successfully disrupted 5 separate HIV-1 exons (tat_1-2/_rev_1-2/_gp41) providing a pathway for CRISPR-directed HIV-1 cure therapies.