Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

technology and developed an HIV-1 specific gene editing molecule that, for the first time, excised the entire HIV-1 genome between the 5′ -and 3′ -LTRs from the host chromosomes of latently infected cells and protected the cells from re-infection 13,16,17 . The method of excision included use of multiplex specific gRNAs that recognize various regions of the 5′ -and 3′ -LTR DNA sequences and the Cas9 endonuclease, which introduces breaks on double-stranded DNA at the sites that are complementary to the gRNAs 16,17 . After removal of viral DNA, the residual cellular DNA re-joins by cellular DNA repair 13,14,16,17 . CRISPR/Cas9 gene editing using a multiplex of different gRNAs that introduce InDel mutations as well as excision of segments of the viral DNA has also been utilized by several other laboratories 18,19 . The use of a multiplex of gRNAs for editing the HIV-1 genome by CRISPR technology is particularly critical in order to alleviate any concern related to the emergence of resistant virus against the initial gRNA treatment. In addition to CRISPR/Cas9 technology, more recently, recombinase based procedures have been developed with the ability to edit the HIV-1 DNA sequence from the host genome 20 .
In the studies presented here, we have refined our gene editing technique and have developed a new strategy that allows conditional activation of the CRISPR/Cas9 at an early stage of viral reactivation by the HIV-1 transcriptional activator, Tat. This new strategy permanently ablates virus replication prior to productive viral replication by removing a segment of the viral gene spanning the viral promoter and/or the viral coding sequence. Further, this strategy will alleviate any concerns due to unforeseen complications that may arise by unnecessary and persistent expression of Cas9 at high levels in cells.

Results
We placed the coding DNA sequence corresponding to the Cas9 gene in a pX260 expression vector, containing three different segments of the HIV-1 promoter spanning the U3 and R regions of the 5′ -LTR to identify the minimal DNA elements of the viral promoter that remain responsive to Tat, yet lacks the sequences corresponding to gRNAs A and B that are initially used for editing HIV-1 DNA (Fig. 1a). After verification of our cloning strategy by DNA sequencing of each construct, expression of Cas9 by each vector and the level of their response to Tat was examined in TZM-bl cells co-transfected with pX260-LTR-Cas9 and CMV-Tat. Results from Western blot revealed activation of Cas9 expression by all three constructs including the plasmid encompassing the minimal DNA promoter sequence positioned between − 80 to + 66 (Fig. 1b). A basal level of the minimal promoter (− 80/+ 66) led to low levels of expression of Cas9 in these cells. This was particularly important for our studies as the promoter sequence resides outside of the DNA sequences corresponding to gRNAs A and B (Fig. 1b). Next, a DNA fragment corresponding to LTR (−80/+66) -Cas9 was cloned into a lentiviral vector (LV) and used to transduce TZM-bl cells to assess the effect of Tat protein on the editing of integrated copies of HIV-1 DNA expressing the luciferase reporter gene. Results from PCR amplification of the LTR revealed the detection of 205 bp DNA fragment in cells expressing gRNAs A and B and Tat protein (Fig. 1c, compare lanes 1-5 to lanes [6][7][8]. The position of the primers used for PCR amplification and the expected amplicons are illustrated in Supplemental Fig. 1. Expression of Cas9, Tat and α -tubulin (control for equal loading) are shown in Fig. 1d.
Next, we examined the impact of the viral DNA excision on viral promoter activity by luciferase assay. Results show a gradual decrease in luciferase activity upon activation of Cas9 by Tat, corroborating the results from DNA assay, indicating that the cleavage of DNA causes inhibition of viral promoter activity in these cells (Fig. 1e). In follow-up studies, we investigated the activation of Cas9 upon infection of TZM-bl cells by HIV-1. To this end, the LTR (−80/+66) -Cas9 reporter TZM-bl cells were transduced by LV-gRNAs A/B for 24 hours, after which cells were infected with HIV-1 JRFL or HIV-1 SF162 at three different MOIs. After 48 hours, cells were harvested for evaluating DNA excision by PCR, expression of the integrated promoter sequence by luciferase assay, and expression of Cas9 by Western blot. Results from these experiments show the detection of a post-cleavage 205 bp DNA fragment in cells infected with HIV-1 JRFL and HIV-1 SF162 , indicating that production of Tat by HIV-1 JRFL and HIV-1 SF162 transactivated the LTR (−80/+66) promoter and production of Cas9 in these cells (Fig. 2b). It was also noted that the level of viral DNA cleavage by Cas9 under HIV-1 SF162 infection is less than that seen in HIV-1 JRFL . This difference may result from the variation in the level of infectivity of the cells by these two viral isolates, and/or the different levels of Tat production by these viruses and their potency in stimulating the minimal LTR promoter that initiates Cas9 expression in these cells. Results from luciferase assay revealed significant reduction of luciferase activity in the cells, again verifying the effectiveness of Cas9 activation by Tat, which is produced upon infection by HIV-1 JRFL or HIV-1 SF162 in shutting down the integrated HIV-1 luciferase gene. Results from Western blot showed activation of the truncated LTR promoter, LTR −80/+66 , upon infection of cells with HIV-1 JRFL and HIV-1 SF162 , resulting in the production of Cas9 protein in the cells (Fig. 2a).
In a follow-up experiment, we tested the ability of Tat-mediated activation of the LTR-Cas9 along with gRNAs A/B in eliminating the HIV-1 genome in the human T-lymphocytic cells line, 2D10 21 . These cells harbor integrated copies of a single round HIV-1 NL4-3 in a latent state, whose genome lacks a portion of the Gag and Pol genes and the Nef gene is replaced by a gene encoding the reporter green fluorescent protein (GFP). The enhanced level of Tat protein in these cells and the activation of Cas9 (shown in Fig. 3a) caused editing of the viral LTR upon activation of Cas9 in the cells transduced by LV-gRNAs A/B (Fig. 3b, also see Supplemental Fig. 2, lanes 1-8). Accordingly, a significant decrease in the number of GFP positive cells was detected in the presence of Tat (Fig. 3c), indicating that activation of Tat eliminates the capacity of the promoter in expressing viral DNA, which in turn, causes suppression of GFP in these cells. The DNA sequence corresponding to the position of the gRNAs, excision of the DNA fragment and PCR primers are shown in Supplemental Fig. 3. The basal level of Cas9 expression and viral DNA excision may attribute to the constitutive but lowest expression of Tat in the latent 2D10 cell line.
In light of earlier observations indicating the ability of PMA and/or TSA in stimulating integrated copies of proviral DNA in 2D10 cells 21 , we sought to assess the impact of PMA and TSA on the activation of Cas9 in a latently infected T-cell model. As seen in Fig. 4a, treatment of 2D10 cells with PMA and TSA, singly or in combination, increased the level of Cas9 expression. In a parallel experiment, we performed PCR analysis for the detection of LTR DNA and showed a clear increase in the level of viral DNA excision (Fig. 4b  cells, indicative of viral activation, by flow cytometry (Fig. 4c) showed a drastic decrease in the level of viral gene expression. Thus, it is likely that production of Cas9 upon activation of the minimal viral promoter (− 80/+ 66) by either Tat, which is expressed upon reactivation of the silent provirus DNA or by PMA and TSA, leads to editing of the integrated copies of viral DNA and exerts a negative effect on the expression of the latent viral genome in cells containing gRNAs A and B.
In the next series of experiments, we examined the level of HIV-1 replication in Jurkat T-cells containing LTR-Cas9. Cells were transduced with lentivirus vector (LV) expressing gRNAs A and B, and LTR (−80/+66) -Cas9. After 24 hours, the transduced cells were infected with HIV-1 NL4-3-EGFP-P2A-Nef , and after 3 and 5 days, cells were harvested and viral DNAs were tested for the excision of a 190 bp DNA fragment spanning gRNAs A and B target sequences. As shown in Fig. 5 (Panels A and B), infection of cells with HIV-1 led to the appearance of a 205 bp amplicon in day 3 whose intensity was increased at day 5 of infection ( Fig. 5a,b). This observation suggests that, similar to the results shown in Fig. 3, an increase in the level of Tat during the course of HIV-1 infection stimulated LTR-Cas9 expression, and hence, cleavage of LTR DNA. Direct sequencing of the 205 bp band seen in day 5 revealed cleavage sites within the LTR by Cas9/gRNA A and Cas9/gRNA B causing a range of InDel mutations that were detected in the junction of the 5′ and 3′ fusion sites (Fig. 5c). Examination of segments of the viral DNAs corresponding to the 5′ -UTR (nt + 97 to + 235) and envelop (env) gene (nt + 5828 to + 5977), both of which are positioned between the 5′ and 3′ LTRs, showed a substantial decrease in the intensity of a 139 bp and 150 bp amplicons corresponding to the 5′ -UTR and env gene, respectively at day 5 compared to day 3 (Fig. 5d). These observations suggest the excision of a larger DNA fragment of the HIV-1 genome spanning between the 5′ and 3′ LTRs upon cleavage by Cas9/gRNA A (at the 5′ LTR) and Cas9/gRNA B (at the 3′ -LTR) may have also occurred upon treatment of the cells with Cas9 and gRNAs A and B, an event that has been reported previously 16,17 . Quantitative analysis of the results from flow cytometry illustrating expression of the reporter GFP, indicative of viral gene expression, showed substantial inhibition of GFP positive cells (64%) on day 3 and even more on day 5 (84%) and day 8 (88%). The presence of lentivirus harboring genes encoding gRNAs and the marker BFP and expression of GFP in the cells were monitored by fluorescent microscopy and the quality of cell cultures was tested by phase microscopy (Supplemental Figure S4A). Quantitative analysis showed that the total number of cells remained unchanged, indicating that similar to the previous observation 17 , no toxicity  Fig. 3 legend (b). GFP reporter assay, by flow cytometry, and representative scatter plot is shown (c).
is associated with this excision strategy. In accord with results from PCR gel analysis (shown in Fig. 5d), results from qPCR and qRT-PCR showed a significant decrease in the level of viral DNA copy numbers corresponding to the Gag gene, i.e. 55% on day 3 and 84% on day 5 and Gag RNA level 91% on day 3 and 96% on day 5 post infection (Fig. 5f,g). We also performed a similar set of studies in human primary cultures of microglia and astrocytes. Results from these studies showed a significant suppression of viral gene expression and viral DNA presence in HIV-1 infected cells transduced with LVs expressing LTR-Cas9 and gRNAs (shown in Supplemental  Fig. 5). Altogether, these observations provide evidence for the use of novel autoregulatory events by employing viral proteins, including Tat, to initiate the editing strategy using CRISPR/Cas9 by excising the viral genome and permanently suppressing viral replication.

Discussion
Since its discovery in 1985 22,23 , the Tat transactivator protein of HIV-1 has been shown to be a critical regulatory protein due to its role in expression of the viral genome at the transcriptional level and its pathogenic impact on uninfected cells. Mechanistically, Tat associates with the RNA sequence located downstream of the initiation site from transcription (nucleotides + 1 to + 59), the so-called transactivation responsive region or TAR. The association of Tat with TAR triggers a series of molecular and biochemical events leading to the formation of pre-initiation and initiation complexes of transcription in proximity to the transcription start site (nucleotide + 1). This complex includes a series of cellular proteins that have the ability to phosphorylate or acetylate components of the complexes including pTEF and RNA polymerase II, thus facilitating transcriptional elongation of RNA (for review see) 24,25 . In addition, the interaction of Tat with various transcriptional factors including NF-κ B 26 , p300/ CBP and GCN5 [27][28][29] can affect transcription of other viral and cellular genes; all of which contribute to the disease spectrum seen in HIV-1 positive AIDS patients 30 . Tat also plays a major role in the productive replication of latent virus in reservoirs once transcription from the reactivated viral promoter leads to an initial round of viral transcription and Tat production. The unique importance of Tat in HIV-1 replication and the pathogenesis of AIDS, provided a strong rationale for serving as a potential target for drug discovery as well as vaccine development. In fact, several potent inhibitors, some with the ability to interfere with Tat-TAR interaction and others with the capacity to prevent Tat communication with its cellular partners, have shown various degrees of efficacy in affecting HIV-1 replication 31 . The strategy that we utilized in this study was to recruit Tat to stimulate Cas9 expression and promote excision of a segment of the viral genome and permanently ablate HIV-1 gene transcription and replication in cells with productive or latent HIV-1. Here we designed a suicide path for HIV-1 that is triggered by Tat and includes editing of the viral genome using CRISPR/Cas9 technology (illustrated in Fig. 6). According to this pathway, production of Tat in the cells, in addition to stimulating its own promoter with the full-length 5′ -LTR sequence, potentiates expression of Cas9 through the same mechanism by a truncated minimal promoter sequence spanning the GC-rich, TATA box, and TAR (− 80 to + 66) regions. Production of Cas9 and its association with gRNAs designed to target the LTR DNA sequence outside of the (− 80 to + 66) induced InDel mutations within the full-length viral promoter and by excising a segment of the gene, can permanently eradicate HIV-1 in the T-cells. In addition to the expected 417 bp DNA fragment representing the full-length LTR sequence, results from short-range amplification of LTR DNA showed a second DNA fragment of 227 bp in size found only in cells expressing Tat. The 227 bp DNA fragment was generated by joining the residual 5′ -LTR to the remaining 3′ -LTR after cleavage by Cas9/gRNA A at either the 5′ -LTR or the 3′ -LTR. It is also likely that ligation of the remaining DNA fragment from the 5′ -LTR with those from the 3′ -LTR after cleavage by Cas9/gRNA created a new template for gene amplification and the appearance of a 227 bp amplicon. As noted in this study, we aimed to target the sequences positioned between nucleotides − 347 to − 328 and − 143 to − 124 by gRNAs A and B, respectively. In earlier studies we demonstrated the safety of HIV-1 DNA cleavage by targeting these two regions of HIV-1 by CRISPR technology, we reported no off-target effects on the cellular genes by a variety of methods including deep sequencing and bioinformatic studies 17 .
Our results demonstrate that the level of viral DNA excision at the early stage of infection is low and as infection continues, more viral DNA is being edited by Cas9 as evidenced by the results from conventional PCR, flow cytometry as well as Taqman quantitative PCR for detection of viral DNA copy numbers. The low level of viral DNA excision at the early stage of infection, i.e. day 3, may be attributed to the low levels of Tat expression and/or engagement of Cas9 with large numbers of non-integrating viral DNAs that are produced at the early phase of infection by reverse transcription. However, at the later stages, once copies of viral DNA are integrated in the host chromosome, the coordinate utilization of multiplex of gRNAs, in this case gRNA A and B, by Cas9 results in the excision of the viral genome and suppression of viral replication in the cells.
The CRISPR/Cas9 gene editing strategy has received attention in biomedical research in recent years due to its extraordinary ability to edit the genome with precision and high efficiency and its simplicity and flexibility of implementation. However, there are several areas that need close attention. For example, it is important to design the most specific and effective gRNAs to avoid off-target effects. The strategy that we have employed for maximizing specificity and avoiding off-target editing was verified by ultra deep sequencing of the whole genome and various other tests, as described 16,17 . Treatment of the cells by a single RNA may lead to the development of mutant HIV-1 as a result of unfaithful NHEJ repair at the site of cleavage, and potentially lead to the emergence of mutant virus that becomes resistant to the initial single gRNA 32,33 . Employment of multiplex of gRNAs, which, by introducing multiplex double-strand breaks across the HIV-1 genome, leads to the excision of a larger segment of viral DNA from the host genome, alleviating this concern and permanently eliminating any chance for the emergence of replication-competent virus 13,[15][16][17][18][19] . The second issue relates to the controlled expression of Cas9 to avoid the unnecessary persistence of expression of the protein that may non-specifically cause injury to the host genome in the long term and/or induce an immune response. Our strategy for conditional expression of Cas9, by HIV-1 Tat, may provide a novel approach for stimulating the silent gene editing molecule to be expressed and to excise HIV-1 DNA at the early stage of virus reactivation. Indeed, several alternative strategies, including Rev/RRE, can independently or in combination with Tat, can be utilized for controlling CRISPR/Cas9 expression in HIV-1 infected cells.
Stable cell lines and subcloning. TZM-bl cells were plated in 6 well plates at 1 × 10 5 cells/well and transfected using Lipofectamine 2000 reagent (Invitrogen) with 1 ug of pX260-LTR (−80/+66) -Cas9 plasmid. Next day cells were transferred into 100-mm dishes and cultured in the presence of puromycin (Sigma) at concentration 1 ug/ml. After two weeks surviving clones were isolated using cloning cylinders (Corning). Two million Jurkat 2D10 cells were electroporated with 10 ug pX260-LTR (−80/+66) -Cas9 plasmid (Neon System, Invitrogen, 3 times 10 ms/1350 V impulse). Forty-eight hours later medium was replaced with medium containing puromycin 0.5 ug/ ml. After one week selection puromycin was removed and cells were allowed to grow for another week. Next, cells were diluted to a concentration of 10 cells/ml plated in 96 well plates, 50 ul/well and cultured for 2 weeks. Both Scientific RepoRts | 6:31527 | DOI: 10.1038/srep31527 TZM-bl and Jurkat 2D10 pX260-LTR (−80/+66) -Cas9 single cell clones were screened for Cas9-FLAG expression after transfections with control pCMV-empty (pcDNA3.1) or pCMV-Tat plasmids by Western blot. Single cell clones with undetectable/very low level of Cas9 under control conditions and very high levels upon Tat overexpression were expanded and used in further experiments.
Lentivirus packaging. HEK 293T cells were co-transfected using CaPO 4 precipitation method in the presence of chloroquine (50 uM) with packaging lentiviral vectors mixtures at 30 ug total DNA/2. 5 × 10 6 cells/100 mm dish. Next day medium was replaced and 24 and 48 h later supernatants were collected, clarified at 3000 RPM for 10 minutes, 0.45 um filtered and concentrated by ultracentrifugation (2 h, 25000 RPMI, with 20% sucrose cushion). Lentiviral pellets were resuspended in HBSS by gentle agitation overnight, aliquoted and tittered in HEK 293T cells. Lenti-LTR (−80/+66) Cas9-Blast lentivirus was tittered by FLAG immunocytochemistry, pKLV-U6-LTR A/B-PGKpuro2ABFP lentiviruses by BFP fluorescent microscopy. Viral stock. For creation of HIV-1 NL4-3-EFGP-p2A-Nef , we used fusion PCR 35 to amplify the EGFP gene, a P2A self-cleaving peptide 36 , and N-terminal of HIV-1 Nef in frame with HIV-1 splicing acceptor originally for HIV-1 Nef expression. DNA was then cloned into the BamHI and XholI restriction sites of the HIV-1 proviral clone pNL4-3 37 obtained from Dr. Malcolm Martin through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. The self-cleaving P2A peptide from porcine teschovirus-1 between the GFP and Nef allows the expression of HIV-1 Nef in full length 38 .
HIV-1 JRFL and SF162 crude stocks used was prepared from supernatants of PBMCs infected with HIV-1 for 6 days, clarified at 3000 RPM for 10 minutes and 0.45 um filtered. Virus was tittered using Gag p24 ELISA.
In vitro HIV-1 infection. Jurkat cells were infected by spinoculation for 2 h at 2700 RPM, 32 °C in 500 ul inoculum containing 8 ug/ml polybrene then resuspended and left for 4 h, then 500 ul of growth medium was added. The next day cells were washed 3 times with PBS and resuspended in growth medium. For infection of astrocytes and microglial cells, primary human fetal brain cells were transduced/infected by incubation with viral stocks diluted in Opti-MEM medium in the presence of polybrene (8 ug/ml) for 4 h, then 1 ml of growth medium was added for overnight. The next day cells were washed 3 times with PBS and fresh grow medium was added.

HIV-1 DNA detection and quantification. Genomic DNA was isolated from cells using NucleoSpin
Tissue kit (Macherey-Nagel) according to the protocol of the manufacturer. For HIV-1 and β-actin specific PCRs (see Table SI.3), 100 ng of extracted DNA was subjected to PCR using Fail Safe PCR kit and buffer D (Epicentre) under the following PCR conditions: 98 °C 5 minutes, 30 cycles (98 °C 30 s, 55 °C 30 s, 72 °C 30 s), 72 °C 7 minutes and resolved in 2% agarose gel. PCR products were subjected to agarose gel electrophoresis, gel purified, cloned into TA vector (Invitrogen) and send for Sanger sequencing (Genewiz). HIV-1 DNA was quantified using TaqMan qPCR specific for HIV-1 5′ -UTR and Env genes and cellular beta-globin gene as a reference (see Table SI.4). Prior to qPCR, genomic DNA from infected cells was diluted to 10 ng/ul and then 5 ul (= 50 ng) was taken per reaction/well. Reaction mixtures were prepared using Platinum Taq DNA Polymerase (Invitrogen) according simplified procedure from Liszewski 39 . 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 minutes, 45 cycles (98 °C 15 s, 62 °C 30 s with acquisition, 72 °C 1 minute). Reactions were carried out and data analyzed in a LightCycler480 (Roche).
Reverse transcription and PCR. Total RNA was extracted from cells using RNeasy kit (Qiagen) with on column DNAse I digestion. Next 1 ug of RNA was used for M-MLV reverse transcription reactions (Invitrogen). cDNA was diluted and quantified using TaqMan qPCR specific for HIV-1 Gag and Env genes and cellular beta-actin gene as a reference (Table SI.4) under the same protocol like genomic DNA but analyzed using relative quantification mode.

Flow cytometry and cell viability assay. GFP expression in 2D10 cells was quantified in live cells using
Guava EasyCyte Mini flow cytometer (Guava Technologies). Cell viability was assessed using propidium iodide (PI) staining. To 200 ul of live cells 10 5 in suspension PI solution was added to final concentration 10 ug/ml. Samples were incubated for 5 minutes at room temperature in the dark. After incubation, samples were acquired using a Guava EasyCyte Mini flow cytometer. In HIV-1 NL4-3-GFP-P2A-Nef infected Jurkat cells, first cells were fixed for 10 minutes in 2% paraformaldehyde then washed 3 times in PBS. For analysis of GFP in the fixed cells we used Guava EasyCyte mini flow cytometry.

Western-blot.
Whole cell lysates were prepared by incubation of Jurkat cells in TNN buffer [50 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA pH 8, 1x protease inhibitor cocktail for mammalian cells (Sigma)] for 30 minutes on ice then pre-cleared by spinning at top speed for 10 minutes at 4 °C. Fifty micrograms of lysates were denatured in 1x Laemli buffer and separated by SDS-polyacrylamide gel electrophoresis in Tris-glycine buffer followed by transfer onto nitrocellulose membrane (BioRad). The membrane was blocked in 5% milk/PBST for 1 h and then incubated with mouse anti-flag M2 monoclonal antibody (1:1000, Sigma) or mouse anti-α -tubulin monoclonal antibody (1:2000). After washing with PBST, the membranes were incubated with conjugated goat anti-mouse antibody (1:10,000) for 1 h at room temperature. The membrane was scanned and analyzed using an Odyssey Infrared Imaging System (LI-COR Biosciences).