Significance For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

Abstract AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

Infection with HIV-1 is a major public health problem affecting more than 35 million people worldwide (1). Current therapy for controlling HIV-1 infection and impeding AIDS development (highly active antiretroviral therapy; HAART) includes a mixture of compounds that suppress various steps of the viral life cycle (2). HAART profoundly reduces viral replication in cells that support HIV-1 infection and reduces plasma viremia to a minimal level but neither suppresses low-level viral genome expression and replication in tissues nor targets the latently infected cells that serve as a reservoir for HIV-1, including brain macrophages, microglia, and astrocytes, gut-associated lymphoid cells, and others (3, 4). HIV-1 persists in ∼106 cells per patient during HAART, and is linked to comorbidities including heart and renal diseases, osteopenia, and neurological disorders (5). Because current therapies are unable to suppress viral gene transcription from integrated proviral DNA or eliminate the transcriptionally silent proviral genomes, low-level viral protein production by latently infected cells may contribute to multiple illnesses in the aging HIV-1–infected patient population. Supporting this notion, pathogenic viral proteins including transactivator of transcription (Tat) are present in the cerebrospinal fluid of HIV-1–positive patients receiving HAART (6). To prevent viral protein expression and viral reactivation in latently infected host cells, new strategies are thus needed to permanently disable the HIV-1 genome by eradicating large segments of integrated proviral DNA.

Advances in the engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) that can disrupt target genes have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome (7⇓⇓–10). These approaches have been used to disrupt HIV-1 entry coreceptors C-C chemokine receptor 5 (CCR5) or C-C-C chemokine receptor 4 (CXCR4) and proviral DNA-encoding viral proteins (8, 9). CCR5 gene-targeting ZFNs are in phase II clinical trials for HIV-1/AIDS treatment (11). Also, various gene editing technologies have recently been shown to remove the proviral HIV-1 DNA from the host cell genome by targeting its highly conserved 5′ and 3′ long terminal repeats (LTRs) (12, 13). However, introduction of nucleases into cells via these nuclease-based genomic editing approaches remains inefficient and partially selective to remove the entire HIV-1 genome. Thus, the key barrier to their clinical translation is insufficient gene specificity to prevent potential off-target effects (toxicities). To achieve highly specific HIV-1 genome editing, we combined approaches to identify HIV-1 targets while circumventing host off-target effects. The resulting highly specific Cas9-based method proved capable of eradicating integrated HIV-1 DNA with high efficiency from latently infected human “reservoir” cell types, and prevented their infection by HIV-1.

Discussion The Cas9/gRNA technology platform is facile, versatile and improving rapidly (23), and clinical application is anticipated, particularly in the fields of virus infection, genetic diseases, and cancer (9, 28, 29). Here, we found that LTR-directed gRNA/Cas9 eradicates the HIV-1 genome and effectively immunizes target cells against HIV-1 reactivation and infection with high specificity and efficiency. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses. In the current study, we have mainly focused our efforts on myeloid lineage cells (microglia/macrophage), which are the primary cell types that harbor HIV-1 in the brain. However, this proof of concept is certainly applicable to any other cell type, including T-lymphoid cells (Fig. S6) (12, 13), astrocytes, and neural stem cells. Our combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus. In addition to an extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, the key design attributes in our study included: bioinformatic screening using the strictest 12-bp+NGG target selection criteria to exclude off-target human transcriptome or (even rarely) untranslated genomic sites; avoiding transcription factor binding sites within the HIV-1 LTR promoter (potentially conserved in the host genome); selection of LTR-A- and -B-directed, 30-bp protospacer and also precrRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp protospacer-based, chimeric crRNA-tracRNA system (16, 30); and WGS, Sanger sequencing, and SURVEYOR assay, to identify and exclude potential off-target effects. Indeed, the use of newly developed Cas9 double-nicking (23) and RNA-guided FokI nuclease (31, 32) may further assist identification of new targets within the various conserved regions of HIV-1 with reduced off-target effects. More recently, a clinical trial using the ZFN gene editing strategy was launched to disrupt the gene encoding the HIV-1 coreceptor, CCR5 (8, 9, 11). Functional knockout of CCR5 in autologous CD4 T cells of a small cohort of patients revealed that in one out of four enrolled subjects, the viral load remained undetectable at the time of treatment (33). Similarly, TALEN and Cas9 have been tested experimentally for efficient disruption of CCR5 and CXCR4 (9, 28, 34⇓⇓–37); therefore, taking them into consideration for clinical trials is anticipated. Whether or not the strategies targeting HIV-1 entry can reach the “sterile” cure of AIDS remains to be seen. Our results show that the HIV-1 Cas9/gRNA system has the ability to target more than one copy of the LTR, which are positioned on different chromosomes, suggesting that this genome-editing system can alter the DNA sequence of HIV-1 in latently infected patient’s cells harboring multiple proviral DNAs. To further ensure high editing efficacy and consistency of our technology, one may consider the most stable region of HIV-1 genome as a target to eradicate HIV-1 in patient samples, which may not harbor only one strain of HIV-1. Alternatively, one may develop personalized treatment modalities based on the data from deep sequencing of the patient-derived viral genome before engineering therapeutic Cas9/gRNA molecules. Our results also demonstrate, for the first time to our knowledge, that Cas9/gRNA genome editing can be used to immunize cells against HIV-1 infection. The preventative vaccination is independent of HIV-1 strain’s diversity because the system targets genomic sequences regardless of how the viruses enter the infected cells. Interestingly, the preexistence of the Cas9/gRNA system in cells leads to a rapid elimination of the new HIV-1 before it integrates into the host genome, just like the way by which the bacteria defense system evolved to combat phage infection (38). Similarly, a gene-editing-based vaccine strategy may be effective in eradicating postintegrated HIV-1 genome and newly packaged proviruses in cells. Therefore, investigation of such HIV-1 vaccination in various latent reservoir cells and animal models with stable expression of Cas9/LTR-gRNAs presents an important next step to assess the ability of Cas9 to eradicate viral reservoirs in vivo. Moreover, in light of recent data illustrating efficient in vitro genome editing using a mixture of Cas9/gRNA and DNA (39⇓⇓–42), one may explore various systems for delivery of Cas9/LTR-gRNA via various routes for immunizing high-risk subjects. Once advanced, one may use gene therapies (viral vector and nanoparticle) and transplantation of autologous Cas9/gRNA-modified bone marrow stem/progenitor cells (43, 44) or inducible pluripotent stem cells for eradicating HIV-1 infection. Here, we demonstrated the high specificity of Cas9/gRNAs in editing HIV-1 target genome. Results from subclone data revealed the strict dependence of genome editing on the presence of both Cas9 and gRNA. Moreover, only one nucleotide mismatch in the designed gRNA target will disable the editing potency. In addition, all four of our designed LTR gRNAs worked well with different cell lines, indicating that the editing is more efficient in the HIV-1 genome than the host cellular genome, wherein not all designed gRNAs are functional, which may be due to different epigenetic regulation, variable genome accessibility, or other reasons. Given the ease and rapidity of Cas9/gRNA development, even if HIV-1 mutations confer resistance to one Cas9/gRNA-based therapy, as described above, HIV-1 variants can be genotyped to enable another personalized therapy for individual patients (10).

Materials and Methods Plasmid Preparation. Vectors containing human Cas9 and gRNA expression cassette, pX260, and pX330 (Addgene) were used to create various constructs, LTR-A, -B, -C, and -D (for details, see SI Materials and Methods). Cell Culture and Stable Cell Lines. TZM-bI reporter and U1 cell lines were obtained from the National Institutes of Health (NIH) AIDS Reagent Program, and CHME5 microglial cells were described previously (17). The detailed procedure for cell growth or preparation of stable cell lines is described in SI Materials and Methods. Immunocytochemistry and Western Blot. Standard methods for immunocytochemical observation of the cells and evaluation of protein expression by Western blot were used as described in detail in SI Materials and Methods. Firefly Luciferase Assay. Cells were lysed 24 h posttreatment using Passive Lysis Buffer (Promega) and assayed with a Luciferase Reporter Gene Assay kit (Promega) according to the manufacturer’s protocol. Luciferase activity was normalized to the number of cells determined by a parallel MTT assay (Vybrant; Invitrogen). p24 ELISA. After infection or reactivation, the levels of HIV-1 viral load in supernatant were quantified by p24 Gag ELISA (Advanced BioScience Laboratories, Inc.) following the manufacturer’s protocol. To assess cell viability upon treatments, MTT assay was performed in parallel according to the manufacturer’s manual (Vybrant; Invitrogen). EGFP Flow Cytometry. Cells were trypsinized, washed with PBS, and fixed in 2% (wt/vol) paraformaldehyde for 10 min at room temperature, then washed twice with PBS and analyzed using a Guava EasyCyte Mini flow cytometer (Guava Technologies). HIV-1 Reporter Virus Preparation and Infections. HEK293T cells were transfected using Lipofectamine 2000 reagent (Invitrogen) with pNL4-3-ΔE-EGFP (NIH AIDS Research and Reference Reagent Program). After 48 h, the supernatant was collected, 0.45-μm filtered and titered in HeLa cells using EGFP as an infection marker. For viral infection, stable Cas9/gRNA TZM-bI cells were incubated 2 h with diluted viral stock, and then washed twice with PBS. At 2 and 4 d postinfection, cells were collected, fixed, and analyzed by flow cytometry for EGFP expression, or genomic DNA purification was performed for PCR and WGS. Genomic DNA Amplification, PCR, TA Cloning, Sanger Sequencing, and GenomeWalker Link PCR. Standard methods for DNA manipulation for cloning and sequencing were used (see SI Materials and Methods). For identification of the integration sites of HIV-1, we used a Lenti-X integration site analysis kit as detailed in SI Materials and Methods. Some PCR products were used for restriction fragment length polymorphism analysis. Equal amounts of the PCR products were digested with BsaJI. Digested DNA was separated on an ethidium bromide-contained agarose gel [2% (wt/vol)]. For sequencing, PCR products were cloned using a TA Cloning Kit Dual Promoter with pCRII vector (Invitrogen). The insert was confirmed by digestion with EcoRI, and positive clones were sent to Genewiz for Sanger sequencing. SURVEYOR Assay. The presence of mutations in PCR products was examined using a SURVEYOR Mutation Detection Kit (Transgenomic) according to the protocol from the manufacturer. Briefly, heterogeneous PCR product was denatured for 10 min in 95 °C and hybridized by gradual cooling using a thermocycler. Next, 300 ng of hybridized DNA (9 μL) was subjected to digestion with 0.25 μL of SURVEYOR Nuclease in the presence of 0.25 μL SURVEYOR Enhancer S and 15 mM MgCl 2 for 4 h at 42 °C. Then, Stop Solution was added and samples were resolved in 2% (wt/vol) agarose gel together with equal amounts of undigested PCR product controls. Selection of LTR Target Sites, WGS, Bioinformatics, and Statistical Analysis. We used Jack Lin's CRISPR/Cas9 gRNA finder tool for initial identification of potential target sites within the LTR. Detailed WGS, bioinformatic, and statistical analyses are described in SI Materials and Methods.

Acknowledgments We thank Jessica Otte for technical support; Jennifer Gordon, Shohreh Amini, and Xuebin Qin for helpful comments; and Jeffrey B. Tatro and Cynthia Papaleo for editorial assistance. This work was supported by National Institutes of Health Grants R01MH093271 (to K.K.), R01NS087971 (to W.H. and K.K.), and P30MH092177 (to K.K.).