Cell cultures

Human peripheral blood mononuclear cells (PBMCs; ATCC, NY, USA) were cultured in a complete RPMI-1640 culture medium supplemented with 10% fetal bovine serum (Gibco, NY, USA) and 100 IU/ml IL-2 (STEMCELL, Vancouver, Canada). PBMCs were thawed and incubated for 2–3 days before nucleofection to ensure viability and proper behaviors. THP-1 and Jurkat cells (ATCC, NY, USA) were maintained in an RPMI-1640 medium containing 10% FBS (Thermo Fisher, NY, USA). THP-1 cells (2 × 105/ml) were differentiated into M0 macrophages using 200nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Missouri, USA) for 2 days. HEK293FT cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (Gibco, NY, USA) with 10% fetal bovine serum (Gibco, NY USA). All cells were cultured under a standard cell culture condition (37°C, 5% CO2) in a humidified incubator (Thermo, NY, USA).

Design of CRISPR guide sequences

dCas9-p300 [14] (Addgene# 61357), dCas9-TET1 [15] (Addgene# 167983), dCas9-VPH [17] (Addgene# 158091), and dCas9-VPR [16] (Addgene# 63798) were purchased from Addgene (Watertown, MA, USA). To construct the required sgRNA guide sequence, a pair of annealed oligonucleotides were cloned into a pU6-sgRNA expression cassette (Addgeen# 53188) bearing the sgRNA scaffold backbone and tracrRNA using BbsI. The oligonucleotides were designed based on the target site sequence (20 bp), and they were flanked on the 3’ end by a 3-bp 5’-NGG-3’ PAM sequence. The CRISPR RGEN Tool, Cas-Designer [47], was used to identify the target sequence of sgRNAs. The first nucleotide of the transcribed gRNA was a guanine nucleotide (G) to maximize the U6 promoter activity. The selected sgRNA target sequences had no potential off-target sites of RNA-guided endonucleases within 2-nt mismatches. The pU6-seq primer was used for DNA sequencing to confirm successful guide sequence insertion in the sgRNA. All primers used for designing sgRNAs are listed in Supplementary Table 1.

Lipofectamine 3000 transfection of CRISPR plasmids

One day before plasmid transfection, 1 × 105 HEK293FT cells were seeded in one well (area: 1.9 cm2) of a 24-well plate. To perform the transfection, 500ng of CRISPR/dCas9-based epigenetic modifiers (and transcriptional activators) targeting human TERT promoter were mixed with 1µL of P3000 Reagent and 1.5µL of Lipofectamine 3000 (Invitrogen, NY, USA) in 200µL of Opti-MEM I Reduced Serum Medium (Gibco, NY, USA). The transfection mixture was added to the cell cultures and incubated overnight. On the day ‘1’, the Opti-MEM medium was then changed to the DMEM medium with 10% FBS. On the day ‘2’, some of the transfected cells were harvested for total RNA extraction and qPCR tests. On the day ‘3’, the remaining cells were harvested for the flow cytometry measurement of hTERT.

Nucleofection of CRISPR plasmids

We used the 4D-Nucleofector™ system (Lonza, Basel, Switzerland) and P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza, Basel, Switzerland) to transfect the plasmid into PBMCs and THP-1-derived macrophages. The nucleofection program F1-115 was used. Briefly, 3.6 µl of the Nucleofector™ Supplement was first added to 16.4 µl of the Nucleofector™ Solution. After centrifugation and aspiration, a pellet of the target cells was resuspended using the nucleofection solution mixture in a syringe tube. Plasmid DNAs (total weight: 1000ng) were added to the cells. An equal amount of each plasmid vector was added, i.e., 500ng of epigenetic modifiers (and transcriptional activators), and 500ng of sgRNAs. The cell/DNA mixture was then transferred into the 4D-Nucleocuvette™ system for nucleofection. The transfected cells were added with 160 µl prewarmed medium, followed by transferring them to a well of the 24-well plate.

qPCR analysis

Total RNA was extracted from the cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). The concentration and purity of the total RNAs were then determined with a Biochrom spectrophotometer. The RNAs were harvested with the required purity and quantity and reverse-transcribed using a SuperScript III First-Strand Synthesis System (Invitrogen, NY, USA), according to the manufacturer’s protocol. For qPCR, the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, California, USA) was used to amplify the synthesized cDNA. The mixture for each qPCR reaction contained 10µL of 2X SsoAdvanced Universal SYBR Green Supermix, 0.2µL of 10µM forward primer, 0.2µL of 10µM reverse primer, 2µL of 2.5ng/µL cDNA sample, and 7.6µL of nuclease-free water. The involved primers are listed in Supplementary Table 1. An exon-exon junction primer was also designed to avoid genomic DNA amplification. The qPCR was implemented using a Thermal Cycling (Bio-Rad, California, USA) Connect System. Procedures of the qPCR were carried out as follows: an initial denaturation step at 95 °C for 3 min followed by 40 cycles at 95 °C for 15s and 62 °C for 45s. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for internal normalization. The relative gene expression was calculated using the 2-ΔΔCt method. Technical triplicates for each sample were performed. A melt curve analysis was done at 65–95 °C with 0.5 °C increments and 2–5 s/step for verifying the specificity of the primers and ensuring no primer-dimer formation during the qPCR.

CD3 and hTERT protein staining

A flow cytometer (BD Biosciences, New Jersey, USA) was used to quantify CD3 and hTERT expressions in cells. To measure CD3 expressed in PBMCs, the cells were stained with FITC-conjugated anti-human CD3 (3 µl of 0.5 mg/ml added to 100 µl PBS) (BioLegend, California, USA) for 30 min at 4 °C in the dark, followed by washing twice with PBS. To determine hTERT protein expression levels, cells were firstly fixed in 200µL of 4% PFA (Thermo Fisher, NY, USA) for 20 min at 4 °C. After washing twice with PBS, the cells were permeabilized in 200µL of 0.1% Triton X-100 (Sigma, Missouri, USA) for 30 min. After washing twice with PBS, the cells were stained with FITC-conjugated anti-human hTERT (1 µl of 200 µg/ml to 100 µl PBS) (Santa Cruz, Dallas, Texas, USA) for 1 h at 4 °C, followed by washing with and resuspended with 105 cells in PBS for flow cytometry analysis. FITC signals of the stained cells were then quantified by the flow cytometer. On the other hand, forward scatter (FSC) and side scatter (SSC) signals were used to quantify the cell size and granularity, respectively.

Western blot for hTERT protein detection

20 µg of protein was loaded into each well of SDS-PAGE gel, followed by transferring into a nitrocellulose membrane for Western blots. Primary antibodies (TERT; stan-cruz cat. # sc-393013 and α-GAPDH; Cell Signaling Technology cat. #14C10) were diluted at 1:1000 in TBST + 5% Milk. Secondary α-Rabbit HRP (Sigma-Aldrich cat. #A6154) and α-Mouse HRP (Beyotime cat# A0216) were diluted at 1:5000 in TBST + 5% Milk. Finally, membranes were exposed and imaged with BIO-RAD ChemiDoc MP Imaging Systemafter the addition of ECL (Bio-Rad cat. #170–5060).

Microscopic imaging

Brightfield and fluorescence microscopic images were taken under an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Cell cycle analysis

Cell cycles of Jurkat, immortalized T cells, and PBMC cells were determined through the measurement of DNA contents in each cell using flow cytometry. Briefly, cells were firstly fixed with 70% cold alcohol, followed by treating them with ribonuclease A (50 µl of 100 µg/ml; Sigma) to remove RNAs from the cell (PI; 200 µl from a 50 µg/ml stock solution purchased from Thermo Fisher). Distributions of cells in the G1, S, and G2 cell cycles were quantified according to the stoichiometric of PI stains.

Cell proliferation assays

Cell proliferation assays were carried out with CFSE stains (CFDA-SE (5(6)-carboxyfluorescein diacetate succinimidyl ester, Thermo Fisher). Fluorescence intensity levels in cells (1) immediately after CFSE staining (Io) and (2) 72-hour and 240-hour after staining (In) were measured to determine the proliferation rate. Because cell division can be visualized as a series of generational divisions that each result in halving the fluorescence intensity, the number of generations (n) can be estimated by solving Io= 2nIn. Additionally, to discriminate the viability of the immortalized T cells, annexin V (Thermo Fisher) was used to detect phosphatidylserine expression over apoptotic cells, and PI stains were used to stain intracellular DNAs according to its capability of penetrating via membranes of late apoptotic/dead cells. Cells were resuspended with PBS before staining with annexin V and PI for 30 min at room temperature. The stained cells were washed with PBS before being analyzed with flow cytometry. Cells of the positive group were treated with 10% DMSO and incubated for 12 h.

Karyotype analysis

Gibco KaryoMAX Colcemid Solution (Invitrogen) was added at a final concentration of 0.1 µg/mL for 120 min to mitotic arrest the metaphase chromosome. Cells were trypsinized and then gently resuspended in 5 mL 37℃ of Potassium Chloride Solution, 0.075 M hypotonic solution (Invitrogen) for 15 min for enlarging cells to facilitate the spreading of metaphase chromosomes for karyotyping at 37℃ water baths. Remove the hypotonic solution and add 5–7 mL of the freshly prepared iced fixative solution (methanol and glacial acetic acid 3:1) for 30 min at 4℃, repeat this step two times to fix the cells, cells can maintain the morphology in this fixation buffer for certain days. Finally, three drops (300–500µL) of the fixative solution were added for karyotyping analysis. 20–30 µL of cell suspension was added onto a clean dry ice slide to spread the chromosome. Gibco KaryoMAX Giemsa Stain Solution (Invitrogen) was used for the G-banding of chromosomes for cytogenetic analysis under a phase contrast microscope (100X magnifications). Count the number of chromosomes in about 50 cells.

Chromatin immunoprecipitation (CHIP)-qPCR

CHIP-qPCR was used to determine the specificity of CRISPR-based transcription activators on modulating epigenetic marks on the TERT promoter. In this assay, HEK293FT cells were co-transfected with activator plasmid (P300, TET1, VPR, and VPH) and gRNA in 10 cm cell culture dishes. Approximately 3xE6 cells were seeded on each 10 cm cell culture dish. Each treatment group was performed in triplicate. Cells were cross-linked at 37℃ for 10 min at a final concentration of 1% formaldehyde (Sigma F8775-500ML) and then the reaction was stopped by the addition of glycine to a final concentration of 125mM for 5 min at RT. Cells were then washed by PBS added with PMSF (proteinase inhibitor, Beyotime ST507-10 ml) and then harvested by cell scraper. Cells were resuspended in the SDS lysis Buffer.

Chromatin was fragmented into an average of 200–600 bp by a sonicator (Shanghai YETO, JY92-IIN). We run the 3% Agarose electrophoresis to measure the chromatin size. 10% of the total chromatin from each lysate was used as Input control. 1ug of each antibody was utilized in each CHIP experiment. The positive group used was the anti-RNA polymerase II antibody (Biolegend, 904004), while the negative group used was anti-mouse IgG (Abcam, ab18413). Anti-human H3K27ac antibody (diagenode C15410174-10ug), anti-human TET1 Antibody (Active Motif, 61444-10ul), anti-c-Myc antibody (Abcam, ab32072), and anti-HSF1 antibody (Abcam, ab52757) were utilized to detect P300, TET1, VPR and VPH activator occupancy on TERT promoter, respectively. All the antibodies were cultured with sonicated chromatin overnight at 4℃, followed by adding Protein A/G Magnetic Beads and culture for another hour. Then, the immunoprecipitated protein-DNA complex was washed by Low, high salt immune complex wash buffer, LiCl immune complex wash buffer, and TE wash buffer. 5 M NaCl, 0.5 M EDTA, 1 M Tris, and proteinase K for 4 h were added to reverse the cross-linking process. GeneJET PCR purification kit (Thermo, K0701) was finally used to recover and purify the DNA. DNA concentration was quantified by Nanodrop with 50ug/ml. Finally, 10ng DNA was used in subsequent qPCR detection. Primer sequences for CHIP-qPCR analysis were listed in the supplementary table.

Telomerase activity

The telomere Repeat Expansion Assay (TRAP) was used to measure the telomerase activity in cells using the TRAPEZE® RT Telomerase Detection Kit (Millipore). Briefly, cells were lysed using 200 µl CHAPS solution and incubated on ice for 30 min. After 20 min centrifugation with 12000xg, 160 µl supernatant was collected for measurement of telomerase activity. BCA kit (Thermo A55864) was used to quantify the protein concentration. Real-time quantitative PCR was performed using a Thermal Cycling (Bio-Rad, California, USA) Connect System. All samples were run in triplicate using the master mix provided with the kit and AccuStart II Taq DNA polymerase (Quanta Bioscience, cat. 733–2258). An equivalent of 1.5 µg protein was used for each reaction. Cycling parameters were 30 °C for 30 min, then 95 °C for 2 min, followed by 45 cycles of 94 °C for 15s, 59 °C for 60s and 45 °C for 10s where fluorescence readings were taken. Finally, data was collected and analyzed by RotorGene Q analysis software using a standard curve prepared from an artificial template provided with the kit. The controls used include positive and negative hTERT control, non-template control, and heat-inactivated control.

Cellular senescence characterization

Senscence-associated β-galactosidase (SA-β-Gal) is an established biomarker associated with cellular aging. Therefore, the Senescence β-Galactosidase Staining Kit (beyotime, cat. C0602) was used to confirm senescent cells. Senescent cells were characterized by blue dye upon catalysis activity of β-Galactosidase on X-gal substrate. We also quantified the expression of senescence-associated beta-galactosidase by flow cytometer by Cell Meter™ Cellular Senescence Activity Assay Kit (AAT Bioquest, cat.23007), according to the manufacturer’s protocol. Senescent cells also were characterized by immunofluorescence analysis of the most well-established senescence marker p21. Cells were fixed with 4% paraformaldehyde and permeabilized samples with 0.1% triton x-100, followed by the blocking step with 5% BSA (sigma, cat.9048468). P21 Primary antibody (2 µg to 1 ml PBS, Abcam, cat.109520) and Alexa Fluor 647 goat anti-mouse lgG (2 µg to 1 ml PBS, biolegend, cat.405322) were utilized to stain cells. The fluorescent images were captured by a confocal microscope (TCS-SP8, Leica Microsystems, Wetzlar, Germany).

Statistics and reproducibility

Significant differences in continuous variables among subject groups were confirmed by ANOVA. All p-values were two-tailed. The statistical significance levels are indicated as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). Each data point is the average level of > 3 repeated measurements.