The CasRx, (pXR001; Addgene #109049), dCasRx (PXR002; Addgene #109050) plasmids, gRNA expression vectors (pXR003, pXR004; Addgene #109053 and #109054), Vesicular Stomatitis Virus glycoprotein (VSV-G) envelope expression vector (pMD2.G; Addgene #12259), lentiviral packaging plasmid (psPax2; Addgene # 12260), iRFP670 fluorescent reporter vector (piRFP670-N1; Addgene#79987) and pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene #67974) were obtained from the Addgene repository (Watertown, MA, USA).
Construction of pBR43IeG-nef+-iRFP670 HIV vector
The open reading frame (ORF) sequence of iRFP670 amplified from piRFP670-N1 vector by polymerase chain reaction (PCR) using Q5® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and primers encoding NcoI and XmaI digestion sites (Supplementary Table S1). The PCR product and pBR43IeG-nef+ (NIH AIDS reporter clone #11349) were then digested with NcoI and XmaI (New England Biolabs, Ipswich, MA, USA). The digested products were separated by 0.8% agarose gel electrophoresis and purified using the Monarch® DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA). The purified products were ligated using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). The ligated product was transformed into Escherichia coli (E. Coli) Stbl competent cells (New England Biolabs, Ipswich, MA, USA). The clones were picked and screened using colony PCR (Supplementary Table S1). The positive colonies were grown in Terrific Broth (Research Products International, Mount Prospect, IL, USA) containing 300 μg/mL ampicillin at 37 °C. The vector sequence was verified by Sanger sequencing (Genewiz, South Plainfield, NJ, USA).
Construction of pKLV2-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector
The pKLV2-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector was constructed using restriction enzyme digestion. The pre-gRNA cassette was PCR amplified using Q5® High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) to encode MluI and BamHI digestion sites as described in Supplementary Table S2. One microgram of this PCR product and pKLV-U6gRNA(BbsI)-PGKpuro2ABFP was digested with MluI and BamHI (New England Biolabs, Ipswich, MA, USA). The digested products were purified, ligated, transformed into E. coli Stbl competent cells (New England Biolabs, Ipswich, MA, USA), colonies were screened by PCR, positive clones were grown, and the vector sequence was further confirmed by Sanger sequencing as described above. Sanger Sequencing primers are described in Supplementary Table S1.
Construction of pVax-CasRx vector
CasRx-eGFP from pXR001 vector was PCR amplified and cloned into pVax1 (ThermoFisher Scientific, USA) by restriction enzyme cloning. The PCR product was digested with KpnI and XbaI (New England Biolabs, Ipswich, MA, USA) and ligated into pVaxI vector. The expression of CasRx-eGFP was evaluated by transient transfection in HEK293T derived Lenti-X™ cells (Takara Bio, Mountain View, CA, USA) cells, followed by visualization of eGFP by flow cytometry and confirmation of CasRx expression by qPCR (Primers listed in Supplementary Table S1).
Construction of CasRx-NES vector
Sequence from PXR001 vector was PCR amplified with specific oligonucleotide to exclude NLS sequence and add nuclear export signal of mitogen-activated protein kinase (MAPK). The rest of the backbone sequence of PXR001 was amplified in a separate PCR. Two DNA fragments with overlapping ends were prepared by consecutive PCR reactions with Q5 DNA polymerase. These fragments were then directly transformed into E. coli Stbl (New England Biolabs, Ipswich, MA, USA) for in vivo cloning as previously described27. Plasmid DNA extracted from resulting colonies was sequenced to confirm NLS signal removal and NES signal insertion in the CasRx-NES vector.
Single gRNA design and cloning
The single gRNAs were designed to target HIV transcript at highly conserved and siRNA targetable sites using the siVirus software (http://sivirus.rnai.jp/). These gRNAs were then confirmed for >70% genetic conservation with all HIV-1 variants within the Los Alamos National Laboratory HIV database (https://www.hiv.lanl.gov/). Those that match this criterion were cloned into the gRNA expression vector, PXR003 using the golden gate assembly. Primers used for gRNA are listed in Supplementary Table S2. 10 µM of each gRNA primer pair was phosphorylated and annealed in a 10 µL reaction containing 1 µL of 10x T4 DNA Ligase Buffer, 0.5 µL T4 polynucleotide kinase, and distilled water. Phosphorylation was carried at 37°C for 30 minutes. The primer pairs were annealed by increasing reaction temperature to 95°C for 5 minutes and allowing the reaction to cool down to 25 °C at a rate of 5°C/minute. The phosphorylated and annealed gRNAs were cloned into PXR003 using a Golder gate cloning reaction. Each reaction contained 1µM annealed crRNA guide oligonucleotides, 25 ng of PXR003, 0.5 µL BbsI (10U/ µL), 0.5 µL T4 DNA Ligase (400 U/ µL), 1 uL of 10x T4 DNA Ligase Buffer, and distilled water in total 10µl. The golden gate amplification was carried out in 30 repeating cycles of 37°C for 5 minutes and 23°C for 5 minutes. The golden gate mix was transformed into E. coli NEB Stbl competent cells (New England Biolabs, Ipswich, MA, USA) and sequenced with U6 promoter primer listed in Supplementary Table S2.
Four gRNAs, targeting four distant sites of HIV-1 genome or non-targeting (NT), along with direct repeat sequences, were collinearly arranged. The entire sequence was commercially synthesized (Genewiz, South Plainfield, NJ, USA) and cloned into pKLV-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP vector using the golden gate assembly with some modifications. The modified 10 µL golden gate reaction contained 50 ng of synthesized polygRNA fragment, 25 ng of the pKLV-U6-CasRx-(pre-gRNA)-PGKpuro2ABFP, 0.5 µL BbsI (10U/µL), 0.5 µL T4 DNA Ligase (400 U/ µL), 1 µL of 10x T4 DNA Ligase Buffer, and distilled water.
HEK293T derived Lenti-X™ cells (Takara Bio, Mountain View, CA, USA) and TZM-bl cells (ARP-8129; NIH AIDS Reagent Program) 28 were cultured in Dulbecco’s Modified Eagle Medium with high glucose, sodium pyruvate, and two mM L-Glutamine (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, USA) and antibiotic-antimycotic solution (ThermoFisher Scientific, USA). Cells were passaged to maintain <80% confluence. Jurkat Clone E6-1 (TIB-152™; ATCC®, Manassas, VA, USA) and HIV latency model J1.1 cells (ARP-1340; NIH AIDS Reagents Program) 29 were cultured in RPMI-1640 media supplemented with 2mM L-Glutamine, 10% bovine calf serum, and 1× antibiotic-antimycotic (Thermo Fisher Scientific). Primary CD4+T cells were isolated from peripheral blood lymphocytes (commercially available single-donor buffy coats from Innovative research https://www.innov-research.com/) and activated with Immunocult™ human CD3/CD28 T cell activator (Stemcell Technologies, Vancouver, Canada), in the RPMI-1640 media supplemented with 2mM L-Glutamine, 10% bovine calf serum, and 1× antibiotic-antimycotic (ThermoFisher Scientific, USA) and human recombinant IL-2 (Stemcell Technologies, Vancouver, Canada). Activated CD4+ T cells were transfected with pVax-CasRx and gRNA plasmids by nucleofection using Lonza 4d nucleofector. Eighteen hours after nucleofection, the cells were infected with HIV-iRFP virus particles. Virus replication was measured by flow cytometry after 48 hours of infection.
Production of virus
The pBR43IeG-nef+-iRFP670 vector was transfected either by itself or with VSV-G envelop plasmid (PMD2.G) into Lenti-X™ cells using Transit-X2 (Mirus Bio, LLC) to make XR-4 tropic or VSV-G pseudotyped HIV-iRFP virus respectively. Antiviral supernatant was harvested 48 and 72 hours following transfection, centrifuged at 500g for 10 minutes at 4°C to remove cellular debris, and concentrated 10-fold using Lenti-X concentrator (Takara Bio, Mountain View, CA, USA). CasRx-GFP lentiviral particles were produced by co-transfecting plasmids encoding CasRx-GFP (PXR001; Addgene) with VSV-G envelope (pMD2.G; Addgene) and packaging (psPAX2; Addgene) into Lenti-X™ cells. Culture supernatants were harvested 48 hours post-transfection, clarified cell debris by centrifugation at 500g for 10 minutes at 4°C, and concentrated 10-fold using Lenti-X concentrator (Takara Bio, Mountain View, CA, USA). To obtain cells with stable CasRx-GFP expression, Lenti-X™ cells were transduced by spinoculation at 800g for 4 hours with CasRx-GFP lentivirus in growth media containing 8 µg/mL polybrene. Forty-eight hours after transduction, the top 5% cells expressing the highest levels of CasRx-GFP were sorted using BD FACS ARIA II (BD Bioscience, USA). The CasRx-GFP expressing cells were expanded and screened for CasRx expression using qPCR (primers listed in Supplementary Table S1).
CasRx-gRNA transfection and measurement of HIV replication by flow cytometry
Twenty-four hours before transfection, stable CasRx-GFP expressing Lenti-X™ cells (LRx) were plated at ∼80,000 cells per well in a 96 well flat bottom plate and incubated overnight to achieve ∼90% confluency before transfection. Two hundred nanograms (ng) of gRNA plasmids and 100 ng of pBR43IeG-iRFP670-nef+ plasmids were co-transfected per well. Each gRNA was transfected in three wells. Forty-eight hours after transfection, the cells were detached and made into a single cell suspension using Versene. HIV replication was measured using a BD Accuri C6 Flow Cytometer (BD Bioscience, USA) as percent HIV-iRFP670 expressing cells in HIV-guide RNA vs. the non-targeting control (NT) guide RNA transfected cells.
J1.1 cells were transfected with pVax-CasRx-GFP and gRNAs using Lonza 4d nucleofector. Eight hours post-nucleofection, the cells were stimulated with phorbol 12-myristate 13 acetate (PMA) and ionomycin (Cell stimulation cocktail, eBioscience, ThermoFisher Scientific, USA), and cell supernatants were collected after 24 hours.
TZM-bl reporter assay
HIV production in the cell supernatants was measured using TZM-bl reporter cell line. Supernatants were added to the TZM-bl reporter cells, and the cells were incubated for 48 h. Cells were lysed, and luciferase activity was measured using Bright-Glo™ reagent (Promega, Madison, WI, USA) as previously described 30.
Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13 mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.
Competing Interest Statement
The authors have declared no competing interest.