Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models

Materials and reagents

The cell line HEK293T used in the present study was obtained from ATCC cell bank and maintained in DMEM medium (Dulbelcco’s Modified Eagle Medium, 11995073, ThermoFisher, MA, USA) supplemented with 10% of fetal bovine serum (A31608, ThermoFisher, MA, USA). J.Lats 10.6, a lymphocyte cell lineage, and the myeloid strain U1 cells, (derived from U937 myeloid cells chronically infected with an HIV-1 clone) used in the study were kindly provided by Dr. Lucio Gama (Johns Hopking Medical School, MD, USA) and originally obtained from the AIDS reagent program of the National Health Institute of the United States of America (NIH, MD, USA). These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Unless stated otherwise, all cell lines were cultured without antibiotics and kept in a conventional cell culture incubator at 37 °C and 5% CO2 atmosphere. The cells were routinely tested for mycoplasma contamination using MycoAlert® Mycoplasma detection kit (LT07-318, Lonza, FL, USA).

The phorbol esters used to induce the activation of HIV-1 promoter in latent cells were Ingenol B (IngB) (Kyolab—BR) at 1 μM and phorbol 12-myristate 13-acetate (PMA) (P8139, Merck, MO, USA) at 1 μg/μL. Suberoylanilide hydroxamic acid (SAHA—SML0061, Merck, MO, USA) and Panobinosat (Pano) (SML3060, Merck, MO, USA) were used at concentration of 5 μM and 0.15 μM, respectively [22].

The CRISPR vector, pLV hU6-sgRNA hUBC-dSpCas9KRAB-T2a-Puro named here as pdSpCas9Krab (pLV_hU6-sgRNA_hUbC-dSpCas9-KRAB-T2A-PuroR was kindly provived by Dr. Charles Gersbach (Addgene plasmid #71236; http://n2t.net/addgene:71236; RRID: Addgene_71236). In addition, pVSV-G and psPAXv2 plasmids were also acquired through Addgene platform. It is important to note that the Cas9 cloned in the vector was from Streptococcus pyogenes as referenced in a previous paper [23].

sgRNAs and AAVS1 locus selection

To identify and design sgRNAs that would be complementary to the HIV-1 genome, the HIV-1 subtype B sequence, clone HXB2, (accession number AF033819 Los Alamos database) was submitted to the CRISPR Pick tool, available in the Broad Institute genetic disorder platform (GPP). This platform designs and ranks the possible sgRNAs minimizing off targets in human genome by aligning the predicted sgRNAs against the human genome sequence (GRCh38—NCBI RefSeq v.10920210514). The same platform was used to design a negative control sgRNA for adenovirus-associated integration site 1 (AAVS1) locus, commonly used as negative control in double strand brake induced by CRISPR/Cas9 system [24].

This region in human genome is inserted in protein phosphatase 1 regulatory subunit 12C (PPP1R12C) intron 1 and is described as a “safe harbor” which means that any changes in the DNA’s structure in this region will not impact the cell.

Assembly of sgRNAs in the pdSpCas9Krab expression vector

The pdSpCas9Krab vector expresses the fused dCas9 of Streptococcus pyogenes and KRAB domain, both being regulated by the UbC promoter. This vector also expresses a self-cleaving T2A catalytic and a puromycin resistant gene in the same open reading frame (ORF) of dSpCas9 and KRAB. The sgRNA is regulated by the U6 promoter that occurs in the same vector, but in the opposite sense of dSpCas9 ORF (Fig. 1B). To insert the nucleotide sequence for the sgRNA of choice, 1.5 μg of the vector pdCas9KRAB was digested with 10 UI/μL of BsmBI (R0739S, New England Biolabs, MA, USA) following the manufacture’s procedures. The sgRNA protospacer were synthetized as independent oligos by Integrated DNA technologies (USA). After the digestion, lyophilized sgRNAs oligos (Additional file 1) were resuspended in H2O DEPC to make 100 μM stocks. T4 PNK (M0201S, New England Biolabs, MA, USA) was used for the phosphorylation step following to manufacturer’s instructions. Then, the reaction was submitted to 95 °C for 5 min and 5 °C for 1 min to join the two DNA strands. The ligation of the sgRNAs sequences to the digested expression vector was performed using T4 DNA Ligase enzyme (M0202S, New England Biolabs, MA, USA), following manufacturer’s procedures. Chemically competent bacteria JM109-prepared using Mix & Go Competent Cells—JM109 strain kit (T3003, Zymo Research, CA, USA)—were transformed with 2 μL of ligation product using manufacturer’s recommendations. Positive bacteria were selected by plating in LB + 50 μg/ml ampicillin plates kept at 37 °C for 24 h and confirmed by colony PCR followed by electrophoresis on a 2% agarose gel, where fragments of approximately 280 bp were expected (data not shown). These confirmation PCR reactions used the U6 forward primer and the one of sgRNA strand as a reverse primer [LTR (1–5) Reverse].

Fig. 1figure 1

CRISPR sgRNAs constructs repress GFP expression in J.Lat 10.6 cells. A A schematic representation of HIV-1 provirus genome (HXB2) indicating the five predicted sgRNA ligation sites and a illustrative scheme of the used CRISPR system: CRISPR sgRNA, dCas9 and KRAB domain B Schematic representation of vector construct (pdSpCas9KRAB and pdSpCas9Ko/KRAB) C GFP background expression of each previous sgRNA (LTR1-LTR5) transduced untreated control J.Lat 10.6 cells. D GFP expression of each sgRNA (LTR1-LTR5) transduced J.Lat 10.6 cells treated with 1 µM PMA. E GFP expression of each sgRNA (LTR1-LTR5) transduced J.Lat 10.6 cells treated with 1 µM IngB. The statistical analysis was done using one-way ANOVA comparing mean of NT with AAVS1 and LTR1 to LTR5 (*p < 0.03; **p < 0.0053; ***p < 0.0003; ****p < 0.0001). The mean values of two technical measures of three independent biological experiments are shown

KRAB sequence excision

The sequence of the repressor protein KRAB was removed from pdSpCas9KRAB using 10 UI of NheI (Promega, WI, USA) and the resulting plasmid was relinked with 1 UI of T4 ligase (M1794, PROMEGA, WI, USA), generating the vector pdSpCas9koKRAB. The excision was confirmed by sequencing the plasmid using the BigDye Terminator protocol (ThermoScientific, MA, USA), according to the manufacturer’s recommendations (data not shown).

Lentiviral vectors production

HEK293T cells were seeded at 8.0 × 105 cells/mL per well in 6-well plates, 24 h before transfection using calcium chloride protocol. The LTR1-5sgRNA/dSpCas9KRAB expression vectors were incubated with a mix solution containing 9 μL of 2.5 M CaCl2, 8 μg of total plasmid (3.53 μg of each expression vectors, 1.76 μg of VSV-G and 2.7 μg of psPAX) and H2O DEPC to a final volume of 90 μL. The same volume of 2X Hepes Buffer Saline (HBS) was added for DNA precipitation. The mixture was incubated at room temperature for 15 min and dripped gently into the well, which had the culture medium previously replaced by a 1 mL of Opti-MEM (31985070, ThermoFisher, MA, USA). The transfection solution was kept in contact with cells for 6 h and after this time replaced by 2 ml of complete medium. After two days, the supernatant from each well was collected, filtered with a 0.22 μm filter and the solution containing the lentiviruses was frozen at − 80 ºC until further use.

Transduction of lentiviral vectors in lymphoid and myeloid cells

The cells used for virus transduction with sgRNA/pdSpCas9KRAB construct were J.Lat 10.6 and U1. For this purpose, 2.5 × 105 cells were plated in a 12-well plate in a final volume of 1 mL. Then, 8 μg/mL polybrene (Merck—H9868) and 500 μL of the lentivirus solution was added to the culture medium. The virus and cell solution were then submitted to centrifugation at 1000g for 2 h at room temperature. After 24 h, 0.5 μg/mL of puromycin (P8833, Merck, MO, USA) was added to select transduced cells. The selection of stable transduced cells took approximately two weeks and was confirmed by PCR when 100 bp fragments should be amplified from selected cells. The forward primers were one sgRNA strand and the U6 promoter reverse primer (Additional file 3: Fig. S1).

Measurement of GFP from induced J-Lat 10.6 cells

Non-transduced (NT)—wild type J.Lat 10.6 cells or J.Lat 10.6 cells transduced with empty vector (EP), empty vector knockout KRAB (EPko/KRAB), AAVS1 sgRNA or LTRs sgRNA with and without KRAB cells (LTR or LTRko/KRAB) were plated at a 105 cells/mL, in 24-well plates and separated into three experimental groups: the untreated group, that did not receive the reactivating drug (negative control); the PMA group treated with 1 μg/μL of PMA and the IngB group which received 1 μM of ingenol B. After 24 h cells were centrifuged at 300g for 4 min and resuspended in 100 μL of PBS. Reactivation was quantified by GFP fluorescence intensity using a BD Accuri C6 flow cytometer (BD Biosciences, NJ, USA).

HIV-1 RNA detection in U1 cell line model

The U1 cell activation process was conducted as previously described by Abreu et al. [25]. U1 NT cells and U1 LTRs were plated, in duplicate, at a concentration of 2.0 × 105 per well. Two conditions per cell type were adopted: without treatment or with 1 µM IngB for 48 h. After treatment cells supernatant were frozen at − 80 °C. Before the RTqPCR test, we diluted the supernatant samples by 1000 times. HIV-1 viral load was estimated by HIV-1 Real Time Amplification Kit through m2000 Real Time System (Abbott, IL, USA). The primers target was manufactured by Abbott to the highly conserved region of pol integrase gene region. Primers sequences were not provided.

Statistical analysis

Statistical calculations were performed using one-way or two-tailored unpaired Student T test using Graph Pad Prism version 8.0.0. p values ≤ 0.05 were considered statistically significant. Experiments were performed in three independents biological replicates with two technical replicates per samples. Statistical data analysis of untreated J.Lat 10.6 ko/KRAB cells experiments were performed in two independent biological replicates with two replicates per samples.

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