Cell culture reagents

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and ciprofloxacin were purchased from Sigma-Aldrich, St. Louis, MO. Diethyl ether, endotoxin-free water, gentamicin, acetonitrile (ACN), methanol, KH 2 PO 4 , bovine serum albumin (BSA), Triton X-100, LC-MS-grade water, and TRIzol reagent were purchased from Fisher Scientific, San Diego, CA. The TZM-bl reporter cell line (AIDS Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD) and HEK-293T cells (the American Type Culture Collection (ATCC), Manassas, VA) were cultured in high glucose DMEM supplemented with 10% FBS and gentamicin (10 µg/ml). Jurkat (Clone E6–1, TIB-152™) cells were purchased from ATCC and cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% FBS and gentamicin (10 µg/ml) (Sigma-Aldrich, St. Louis, MO). PBMCs were isolated from leukopaks by gradient centrifugation on Ficoll-Paque for 30 minutes at 600 g. PBMCs collected from the buffy coat were stimulated with PHA (5 µg/ml) for 24 h in RPMI with 10% FBS and gentamicin (10 µg/ml) supplemented with human recombinant interleukin-2 (rIL-2) at a concentration of 30 ng/ml ((STEMCELL Technologies, Seattle, WA). Fresh media was exchanged every 2–3 days.

Cell culture HIV-1 infection

HEK-293T cells were transfected using CaPO 4 precipitation method in the presence of chloroquine (50 µM) with 30 µg of pNL 4–3 -EGFP-P2A-Nef plasmid22 /2.5 × 106 cells/100 mm dish. Next day, media were replaced; and 24 and 48 h later supernatants were collected, clarified at 1400 g for 10 minutes, filtered through 0.45 µm filter, and concentrated by ultracentrifugation for 2 h with 20% sucrose cushion. Viral pellets were resuspended in Hank’s Basic Salt Solution (HBSS) by gentle agitation overnight, aliquoted, and tittered in Jurkat cells by FACS for GFP expression. Jurkat cells were infected by spinoculation for 1.5 h, 32 °C in 500 µl inoculum containing 8 µg/ml polybrene then resuspended and left for 4 h then 500 µl of growth medium was added. Next day, cells were washed 3 times with phosphate-buffered saline (PBS) and re-suspended in growth medium.

Design of gRNA, construction of CRISPR-Cas9 expression plasmid and the AAV 9 vector

Bioinformatics design and cloning of LTR1 and GagD gRNAs into AAV-CMV-saCas9 vector was previously described19,23. Briefly, the Broad Institute gRNA designer tool (https://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design) was used to screen HIV-1 NL4–3 or ADA sequences for possible gRNA protospacer regions followed by saCas9 specific PAM: NGGRR(N). A pair of gRNAs showing the best predicted on-target (in HIV-1 genome) and the lowest off-target (in human genome) activities was selected: one targeting the HIV-1 LTR promoter region and the other targeting gag gene. The sequences of respective LTR1 and GagD gRNAs plus PAM were further cross referenced with Los Alamos HIV sequence database confirming high levels of conservation (>90%) across the HIV-1 sequences. Next, pair of oligonucleotides for each target site with 5′-CACC and 3′-AAAC Bsa1 overhangs was obtained from Integrated DNA Technologies (IDT, Coralville, Iowa, Table S1), annealed, phosphorylated, and ligated into BsaI digested, dephosphorylated pX601-AAV-CMV:NLS-saCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (a gift from Feng Zhang via Addgene) (61591; Addgene). For multiplex gRNA cloning, the U6-LTR1-gRNAscaffold cassette from pX601-CMV-saCas9-LTR1 was amplified using T795/T796 primers (Table S1) and cloned using In-Fusion HD Cloning Kit (Clontech, Mountain View, CA) into EcoRI and KpnI linearized pX601-CMV-saCas9-GagD plasmid resulting in pX601-CMV-saCas9-LTR1-GagD AAV delivery vector. Finally, sequence verified plasmid was sent for packaging into AAV-9 serotype (Vigene Biosciences Inc., Milton Park Abingdon, UK). AAV 9 was chosen as the vector for CRISPR-Cas9 delivery for its robust transduction efficiencies in multiple tissues including the central nervous system as significant putative reservoirs for HIV-1. The notion was to permit efficient AAV entry into all putative HIV-1 target tissues including the brain.

HIV-1 infection of CD34 + humanized mice

NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were obtained from the Jackson Laboratories, Bar Harbor, ME and bred under specific pathogen-free conditions at the University of Nebraska Medical Center (UNMC) in accordance with the ethical guidelines set forth by the National Institutes of Health for care of laboratory animals. CD34 + HSC were enriched from human cord blood or fetal liver cells using immune-magnetic beads (CD34 + selection kit; Miltenyi Biotec Inc., Auburn, CA, USA). CD34 + cell purity was >90% by flow cytometry. Cells were transplanted into newborn mice irradiated at 1 Gy using a RS‐2000 × ‐Ray Irradiator (Rad Source Technologies, Buford, GA). Cells were transplanted by intrahepatic (i.h.) injection of 50,000 cells/mouse in 20 μl phosphate-buffered saline (PBS) with a 30-gauge needle. The experiments shown in Fig. 2–6 were from human fetal liver cells were isolated from a single donor. In the study described in Figs. 7 and 8, cord blood-derived HSC were obtained from two donors. Mice from a single donor were used for all dual treatment mice. Humanization of the animals was affirmed by flow cytometry31,60 for the presence of human CD45 and CD3 positive blood immune cells, as shown in Fig. 4. At 18 weeks of age, 25 NSG-hu mice were infected intraperitoneally (i.p.) with HIV-1 NL4–3 32,36 at 104 tissue culture infective dose 50 (TCID 50 )/ml and sacrificed at days 1, 3, 7, and 14; n = 5 at each time point. Five control-uninfected animals were included in all test evaluations. Levels of viral RNA copies/ml were analyzed with the automated COBAS Ampliprep System V2.0/Taqman-48 system (Roche Molecular Diagnostics, Basel, Switzerland)30,31. For this assay, 100 μl of mouse serum was diluted to 1 ml with sterile filtered normal human serum. The detection limit of the assay after dilution is 200 viral RNA copies/ml. Although the eclipse phase for viral infection in humans remains variable61, the viral loads and CD4 + T cell depletion levels observed in our infected humanized mice are in point of fact reflective of the disease course in an infected human host. Indeed, only after weeks of infection we do observe significant cell loss12,17,29,37,50. These findings can be viewed as an affirmation of the model including CD4 + T cell timed-restorations seen after ART as is seen in humans.

Nanoformulated antiretroviral drugs

DTG, 3TC and ABC were generous gifts from ViiV Healthcare, Research Triangle Park, NC. RPV was purchased from Hangzhou Bingo Chemical Co., Ltd, Hangzhou, China. Antiretroviral prodrugs and their polymer encasements were performed as previously described12,13,14. Myristoylated modifications for DTG, 3TC, and ABC were made (referred to as MDTG, M3TC, and MABC) to enhance the incorporation into poloxamer 407 (P407) nanoparticles, while RPV was encased solely by poloxamer 338 (P338) in unmodified form using high pressure homogenization to form crystalline nanoformulated drugs. Particle size, polydispersity index, and zeta potential were determined by dynamic light scattering using a Malvern Nano-ZS (Malvern, Worcestershire, UK)49. Final drug concentrations in the nanoformulation suspensions and injection solutions were determined by HPLC-UV/Vis and UPLC-MS/MS. A 40–50 μl volume for each nanoformulation combination (NMDTG/NRPV and NM3TC/NMABC) was administered by intramuscular (IM) injection in opposing thigh muscles of the mice.

Antibodies

For flow cytometric analysis, we used a panel of antibodies (all from BD Biosciences, San Jose, CA) comprised of FITC-conjugated mouse anti-human CD45 (catalog #555482), Alexa Fluor 700-conjugated mouse anti-human CD3 (catalog #557943), APC-conjugated mouse anti-human CD4 (catalog #555349), and BV421-conjugated mouse anti-human CD8 (catalog #562428), PE-conjugated mouse anti-human CD14 (catalog #555398), and PE-Cy5-conjugated mouse anti-human CD19 (catalog #555414) antibodies. For immunohistochemical staining, we used monoclonal mouse anti-human HIV-1p24 (clone Kal-1, M0857, Dako, 1:10), monoclonal mouse anti-human leukocyte antigen (HLA-DR; clone CR3/43, Dako, 1:100), and the polymer-based HRP-conjugated anti-mouse EnVision + secondary antibodies were purchased from Dako (Carpinteria, CA). Peripheral blood was collected from the submandibular vein into ethylenediaminetetraacetic acid (EDTA)-coated tubes or by cardiac puncture at the study end. Blood leukocytes were tested for human pan-CD45, CD3, CD4, CD8, CD14, and CD19 markers as six-color combinations using LSR-II FACS analyzer (BD Biosciences). Antibodies and isotype controls were obtained from BD Pharmingen, San Diego, CA, and staining was analyzed with a FlowJo (BD Immunocytometry Systems, Mountain View, CA). The gating strategy is shown in supplementary fig. 20. Results were expressed as percentages of total number of gated lymphocytes. The percentages of CD4 and CD8 positive cells were obtained from human CD3 + gate17. We used absolute counts of human CD45 + cells to normalize each of the human cell data sets. Equivalent numbers of total blood cells/mouse were used at each time point.

Immunohistochemistry (IHC) examinations

Spleen, lung, liver, and lymph nodes were perfused with PBS followed by 4% paraformaldehyde and then post-fixed overnight and processed for paraffin embedding. Five-micron thick sections were cut from the paraffin blocks, mounted on glass slides, and labeled with mouse monoclonal antibodies (Dako) for HLA-DQ/DP/DR (clone CR3/43, 1:100) and HIV-1p24 (1:10). The polymer-based HRP-conjugated anti-mouse Dako EnVision system was used as a secondary detection reagent and developed with 3,3′-diaminobenzidine (DAB). All paraffin-embedded sections were counterstained with Mayer’s hematoxylin. Deletion of primary antibodies or using mouse IgG served as controls. Images were obtained with a Nikon DS-Fi1 camera fixed to a Nikon Eclipse E800 (Nikon Instruments, Melville, NY) using NIS-Elements F 3.0 software.

Nucleic acid extractions and qPCR assays

In studies presented in Fig. 2–8, total viral nucleic acids (RNA and DNA) were extracted from the spleen, bone marrow cells, lung, gut, liver, kidney, and brain using a Qiagen Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total cellular DNA obtained from the HIV-1 infected cell line ACH2 served as a positive control and standards, while human genomic DNA obtained from uninfected NSG-hu mice served as a negative control. Cell-associated HIV-1 RNA and DNA were quantified by real-time qPCR and droplet digital PCR (ddPCR) assays. Because of extremely low numbers of latently-infected human cells in HIV-infected NSG-hu mice after long-term ART, detection of total HIV-1 DNA, required two rounds of PCR amplification. The first round of PCR was performed on a conventional PCR machine (T100 Thermal Cycler, Biorad, CA) in 25 μl of PCR reaction mix containing 500 ng of template and 50 ng each of both primers annealing to HIV-1 gag region and the reaction conditions are as follows: 94 °C for 3 min, followed by 15 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The product of the first PCR was subsequently used as a template in the second semi-nested real-time PCR amplification performed on the ABI Step One Plus real-time PCR machine (Applied Biosystems, Foster City, CA) using TaqMan detection probe and primers30. Two μl of the first PCR product was diluted to 50 μl with PCR master mix containing two primers at 0.2 μM each and 0.2 µM TaqMan dual-labeled fluorescent probe. Real-time PCR settings were as follows: 50 °C for 2 min, then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The amplicon sizes are 221 bp for the first round of PCR and 83 bp for the second round (real-time) PCR. DNA extracted from ACH2 cells containing one integrated copy of HIV-1 per cell was used as standard in serial 10-fold dilutions with HIV copy numbers ranging from 101 to 105 DNA copies/reaction36,37. Integrated DNA (iDNA) provirus was quantified using an adapted Alu-gag PCR assay as described by Agosto et al.62 with modifications for the second round of PCR, following prior published methods63. Briefly, samples underwent a first-round PCR amplification (95 °C for 2 min; 20 cycles of 95 °C for 15 s, 50 °C for 15 s, and 72 °C for 150 s) using 100 nM Alu and 600 nM gag reverse primers. Five μl of the first-round product were amplified in a nested protocol using the assay for HIV-1 gag gene (second PCR primers and probe), as described above. First-round PCR included 3 replicates using only gag reverse primer (gag only) to serve as background un-integrated control. Integration levels per cell were calculated by subtracting gag-only signals from the Alu-gag quantification. Semi-nested real-time RT-PCR on HIV-1 RNA was performed as described36,37. The eluted cellular RNA was first subjected to DNase treatment to remove HIV-1 DNA to avoid the interference with the quantitation. For reverse transcription assay, random hexamers were used as primers and SuperScript III (Invitrogen, MA) to synthesize first-strand cDNA at 42 °C for 60 min. cDNA was used for the unspliced (usRNA) assay. Two rounds of PCR were performed under the same PCR conditions as described for total viral DNA. For the usRNA assay, real-time PCR was run for 45 cycles and same primers and fluorescent probe as for the total viral DNA assay were used. Human CD45 species-specific primers and probes were obtained from Thermo-Fisher Scientific (USA) (cat. no. 433182 for Hs0036534_g1).

For viral excision testing, frozen tissues sent to Temple University from the University of Nebraska Medical Center were homogenized using Bullet Blender homogenizer (Next Advance, Averill Park, NY) using bead combinations and settings specific for every tissue according to manufacturer’s protocols. T1 buffer from NucleoSpin Tissue kit (Macherey-Nagel, Duren, Germany) was used for homogenization/initial lysis followed by over-night proteinase K digestion. Extraction of genomic DNA was completed according to the manufacturer’s protocol. For standard PCRs (supplementary Table 1), 500 ng of extracted DNA were subjected to PCR using Fail Safe PCR kit and buffer D (Epicentre, Madison, WI) under the following PCR conditions: 94 °C 5 min, 30 cycles (94 °C 30 s, 55 °C 30 s, 72 °C 30 s), 72 °C 7 min using 1st round primers followed by nested PCR using diluted 1st round PCR reaction. Nested PCR products were subjected to Sanger sequencing directly if only one amplicon population was detected by agarose gel electrophoresis. For multiple amplicons detected, in order to investigate the composition of HIV excision, each amplicon population was separated and purified from an agarose gel electrophoresis and then cloned into TA vector (Invitrogen, Carlsbad, CA). Plasmid DNA containing excised HIV amplicon was purified from each bacterial colony for Sanger sequencing (Genewiz, South Plainfield, NJ). HIV-1 DNA was quantified using TaqMan qPCR specific for HIV-1 pol and env genes and cellular beta-globin gene as a reference (supplementary Table 1). Prior to qPCR, genomic DNA was diluted to 10 ng/µl and then 5 µl (50 ng) were taken per reaction/well. Reaction mixtures were prepared using Platinum Taq DNA Polymerase (Invitrogen) according to a simplified procedure64. Standard was prepared from serial dilutions of U1 cells genomic DNA since it contains two single copies of HIV-1 provirus per diploid genome equal to beta-globin gene copy number. qPCR conditions: 98 °C 5 min, 45 cycles (98 °C 5 min, 45 cycles (98 °C 15 s, 60 °C 30 s with acquisition, 72 °C 1 min). Reactions were carried out and data analyzed in a LightCycler96 (Roche, Basel, Switzerland). For RT-PCR, TRIzol reagent (Ambion, Austin, TX) was used for initial RNA extraction followed by clean up using RNeasy kit (Qiagen, Hilden, Germany) with DNAse I digestion in the extraction column. Total 0.5ug of RNA was used for M-MLV reverse transcription (Invitrogen). For gRNA expression screening specific reverse primer (pX601gRNA scaffold/R, supplementary table 1) was used in RT reaction followed by standard PCR using target LTR 1 or GagD sense oligos as forward primers (supplementary table 1) and agarose gel electrophoresis. For checking saCas9 mRNA expression oligo-dT primer mix was used in RT and cDNA was subjected to PCR using saCas9 specific primer pairs and β-actin as a reference (supplementary table 1). Sanger sequencing results were analyzed using Clustal Omega (EMBL-EBI) multiple sequence alignment tool and Sequence Scanner Software 2 (Applied Biosystems).

ddPCR for detection of HIV-1 nucleic acids

ddPCR was performed based on the water–oil emulsion droplet technology, using the ddPCR™ Supermix for Probes reagents in the QX200™ Droplet Digital™ PCR system (Bio-Rad Laboratories, Hercules, CA). For quantification of HIV-1 DNA, the eluted cellular DNA was PCR-amplified36,37,63 using Taqman sets targeting the HIV-1 LTRs, gag and pol genes and as a reference mouse or human beta-actin (supplementary Table 1). A total of 100–200 ng DNA from each tissue was used as template for ddPCR amplifications with the same thermal cycling conditions used for real-time q-PCR detection. Data acquisition and analysis were done using QX200 droplet reader and QuantaSoft™ software provided with the instrument. The genomic DNA extracted from a total of 50,000 cells including human and mouse cells was used as template for each ddPCR analysis. The reduction of Gag represents a deletion between 5’LTR and Gag or 5’LTR to 3’LTR, while a reduction in Pol represents the excision between Gag to 3’LTR or 5’LTR to 3’LTR. However, a single LTR will always remain detectable in all three conditions. Thus, we used the ratio of Gag or Pol to LTR to estimate the excision efficiency. For example, in mouse M4349 (Fig. 6e), the ratios of Gag/LTR and Pol/LTR are 19.7% (15 cells with detectable gag out of 76 cells with detectable LTR) and 19.5%, respectively, in the genomic DNA extracted from the spleen of the treated mice. Thus, the excision efficiencies of 5’LTR to Gag and Gag to 3’LTR were estimated to be about 80% for both (100%-19.7% or100% - 19.5%). In the spleen of the same mouse, the AAV9 transduction efficiency was calculated as high as 1.03 AAV vector copies/cell in the total population including both human graft and mouse host cells (Fig. 6g). In another mouse M4346 (Source Data file for Fig. 6e), we demonstrated that the excision occurred mainly in Gag to 3’LTR because the ratio of Pol/LTR is 38.4% while Gag/LTR is 89.5%. Thus, the excision efficiency was estimated at 61.6% in 5’LTR to Gag and 10.5% in Gag to 3’LTR. Nonetheless, the presence of 2 LTRs in an uncut HIV proviral DNA was not considered in order to simplify the estimate.

RNAscope

Viral RNA was detected as single brown dots or cluster of dots in 5 μm thick paraffin embedded spleen and lymph node tissue sections using antisense probe V-HIV1- Clade-B (Cat no 416111) targeting 854–8291 bp of HIV-1 NL4–3 65. Human peptidylprolyl Isomerase B (PPIB) was used as positive control for the spleen tissue analyzed (images were captured at 40-x magnification). All reagents are from Advanced Cellular Diagnostics, Newark, CA.

Viral recovery

PBMCs obtained from leukopaks from HIV-1,2 seronegative donors were stimulated with PHA and IL-2 and co-cultured with human bone marrow or spleen cells recovered from 3 groups of CD34 + HSC- NSG mice that included HIV- 1 infected, infected and LASER ART treated, and LASER ART and AAV 9 -CRISPR-Cas9-treated mice. PBMCs were used in assays after a 3-day treatment maintained in 10% RPMI with 30 U/ml of IL-2 then co-cultured with human bone marrow or spleen cells at concentrations of (1:5)66,67,68. Cells were harvested eight days later for HIV-1 DNA (A) and RNA (B) using semi-nested real-time qPCR assay and supernatant fluids assayed for reverse transcriptase activity for up to day-14. Data are expressed as total HIV-1 DNA (A) or RNA (B) copies/106 human CD45 + cells. One of the two dual-treated animals was tested and confirmed viral sterilization. Viral rescue was observed in other groups of animals tested.

Adoptive transfers

Splenocytes and bone marrow cells (8–10 × 106) were harvested at the time of sacrifice from NSG-hu mice that were HIV-1 ADA infected with and without LASER ART and AAV 9 -CRISPR-Cas9. The cells were adoptively transferred into unmanipulated 18-week old CD34 + HSC-NSG mice. Cell counts and viability tests were determined by both trypan blue and live/dead stains on the TC-20 automated cell counter (Bio-Rad). Cells were injected IP into mice and monitored for an additional 4 weeks. These experiments were performed to cross validate eradication of viral infection that could occur from latent reservoirs and not detected by either qPCR, RNAscope, and ddPCR assays. Viral load was measured from blood samples of the adoptively transferred mice using automated COBAS Ampliprep System V2.0/Taqman-48 system, and immune cell profiles (CD4 and CD8 + T cells by flow cytometry) recorded, in parallel. Residual virus from all humanized mice tissues was examined by qPCR and ddPCR assays. Virus was not detected in plasma or tissues from two adoptively transferred animals (mice M3319 and M3336).

Off-target analyses

TZM-bl cells were plated in 6 well plates at 1 × 105 cells/well and co-transfected using Lipofectamine 2000 reagent (Invitrogen) with 1 µg of control pX601-AAV-CMV:NLS-SaCas9-NLS-3xHA-bGHpA;U6::Bsa1-sgRNA (Addgene catalog #61591) or 1 µg of pX601-LTR1-GagD (20) plasmid together with 0.2 µg of pKLV-U6gRNA(Bbs1)-PGKpuro2ABFP (Addgene catalog #50946) to provide puromycin selection marker. The next day, cells were transferred into 100-mm dishes and cultured in the presence of puromycin (Sigma) at concentration 1 µg/ml. After two weeks, surviving clones were isolated using cloning cylinders (Corning, Corning, NY, USA). Genomic DNA was prepared from each single cell clone and LTR specific PCRs followed by gel purification; TA cloning and Sanger sequencing were performed. The clones showing the presence of on target CRISPR-Cas9 induced InDel mutations at target LTR 1 site in integrated HIV-1 LTR sequence (n = 6) together with two control clones were selected for further in vitro off target analysis. The list of potential OFF target sites in human genome for HIV-1 target LTR 1 and GagD was created using Benchling CRISPR design tool (https://benchling.com/, supplementary table 2). A total of three potential OFF target sites were chosen (the top scorer plus two top gene specific potential off target sites, see supplementary table 2, highlighted in yellow) for PCR based screening in selected single cell clones. The potential OFF target regions were PCR-amplified, cloned into TA vector, and performed Sanger sequencing (3–6 sequences/single cell clone/single OFF target) (supplementary table 3).

The genetic variation analyses among the three treatments were performed through the next generation sequencing (by the Novogene NGS facility) and bioinformatics tools for four sample animals, one animal from the LASER ART, one animal from CRISPR-Cas9- and two no-rebound animals from the LASER ART/CRISPR-Cas9 groups. The main objective was to detect the possible CRISPR-Cas9 off-target sites. Besides this, some genetic variations such as single nucleotide polymorphisms (SNP), insertion-deletions (InDels), structural variants (SVs), and copy number variants (CNVs) were analyzed for those four animals; and the results are in supplementary tables 4–5. After a thorough quality control step, the resulting paired-end short-reads were mapped to the human reference genome (Human_G1K_V37) utilizing Burrows-Wheeler Aligner (BWA) algorithm. For the animals M4356 (CRISPR-Cas9), M4348 and M4349 (LASER ART + CRISPR-Cas9), and M3539 (LASER ART), the 8 coverages were reported to be 92.01%, 91.97%, 92.01%, and 91.92%, while the sequencing depths were 36.08, 63.11, 45.22, and 15.41, respectively.

Excision efficiencies and hierarchal clustering

The excision efficiencies for each animal, tissue, and HIV-1 gene segment were calculated as the ratio of the number of the sequencing-verified PCR product to all members in each group with denoted experimental conditions (i.e. treatments, cells and tissues shown in Figs. 3, 4, 5, 6 and supplementary fig. 4). Defined such that the excision efficiencies can be viewed as frequentist probabilities, i.e. the ratio of the frequency of occurrence of the event of interest to the total number of experimental repeats. This interpretation of excision efficiencies provides the user with a predictive value, as they can be used to set a prior expectation on the success rate of each treatment (LASER ART, CRISPR-Cas9, and LASER ART plus CRISPR-Cas9) in excising the desired segments of HIV-1 gene in the studied tissues and further to relate that to the likelihood of cure. Hierarchical clustering was performed on the efficiency values of truncation events under different treatments and across different animals, tissues, and HIV-1 gene segments. Once the excision efficiencies were calculated under different combinations of experimental conditions, the hierarchical clustering scheme was employed to group the efficiency values into a multilevel cluster tree represented by a dendrogram. This hierarchical clustering heat map may offer a predictive capability for viral elimination. Bioinformatics analysis of human genome sequence data identified human genome sites that could serve as off targets for gRNAs designed for editing HIV-1 DNA. The corresponding efficiency values were listed in heat-map table to make the clusters visually detectable. To this end, three combinations were considered: i) excision probabilities of different HIV-1 segments in 6 different tissues of animals undergoing antiretroviral treatment, CRISPR-Cas9 mediated editing, and the combined treatments; ii) excision probabilities of different segments in different animals under the three treatments; and iii) probabilities of observing at least one positive band for each specified tissue in all animals. Clusters of supplementary figs. 10–16 also include additional conditions of cure and qPCR data to identify which animals experienced complete cure and highest viral genome eradication.

Study Approval

All experimental protocols involving the use of laboratory animals were approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC) ensuring the ethical care and use of laboratory animals in experimental research. Human blood cells were isolated by leukapheresis from HIV-1/2 and hepatitis seronegative donors and were deemed exempt from approval by the Institutional Review Board (IRB) of UNMC. Human CD34 + hematopoietic stem cells were isolated from human fetal liver and umbilical cord blood and are exempt from UNMC IRB approval.

Statistics

The data were analyzed using GraphPad Prism 7.0 software (La Jolla, CA) and presented as the mean ± the standard error of the mean (SEM). Experiments were performed using a minimum of three biologically distinct replicates. Sample sizes were not based on power analyses as the efficiency of HIV-1 elimination was not known nor could it be projected. For comparisons of two groups, Student’s t test (two-tailed) was used. Tissue drug levels, HIV-1 RT activity, HIV-1p24 antigen staining, T-cell populations, viral RNA and DNA, and viral load were analyzed by one-way ANOVA with Bonferroni correction for multiple-comparisons. For studies with multiple time points, two-way factorial ANOVA and Bonferroni’s post-hoc tests for multiple comparisons were performed. Extreme outliers beyond the 99% confidence interval of the mean and 3-fold greater than the SEM were excluded. Significant differences were determined at P < 0.05.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.