
==== Front
Cell Mol Life Sci
Cell Mol Life Sci
Cellular and Molecular Life Sciences: CMLS
1420-682X
1420-9071
Springer International Publishing Cham

39287670
5415
10.1007/s00018-024-05415-9
Original Article
Extended replicative lifespan of primary resting T cells by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators
Huang Siping
Lau Cia-Hin
http://orcid.org/0000-0001-5337-2578
Tin Chung chungtin@cityu.edu.hk

Lam Raymond H. W. rhwlam@cityu.edu.hk

grid.35030.35 0000 0004 1792 6846 Department of Biomedical Engineering, City University of Hong Kong, P6414, Yeung Kin Man Academic Building, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong, SAR China
17 9 2024
17 9 2024
12 2024
81 1 4076 3 2024
24 7 2024
16 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-024-05415-9.

Keywords

CRISPRa
Epigenome editing
Immortalization
Lifespan extension
Longevity
Replicative senescence
Research Grant Council Hong Kong11215619 Lam Raymond H. W. http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 31770920 Lam Raymond H. W. issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

Most mammalian somatic cells have a finite proliferative lifespan due to progressive telomere shortening, largely caused by deficiency of telomerase reverse transcriptase (TERT) [1]. Whereas such natural telomere erosion-induced cellular senescence has its significance in protecting against physiological problems, e.g., cancer and age-related tissue/organ disorders [2, 3]. Cell immortalization can offer a sustainable source of a particular cell type with consistent genetic and phenotypic characteristics for cell research and related medical applications [4]. In the absence of telomerase, telomeres shorten with cell replications, restricting the proliferative capacity and triggering permanent cell cycle arrest known as replicative senescence [5]. On the contrary, ectopic overexpression of TERT transgene was shown as an effective cell immortalization strategy to sufficiently inhibit replicative senescence in the cell [6, 7] and prolong the lifespan in various animal models [8, 9]. In fact, one of the most commonly used approaches to extend the life of primary cells is to introduce exogenous telomerase to the cell by overexpressing the TERT transgene in an episomal form or knocking in the TERT transgene into the genome via retrovirus or lentivirus vector transduction [10]. Compared to other immortalization methods such as overexpressing SV40T antigens or suppressing tumor suppressor genes, exogenous human TERT (hTERT) substantially prolongs replicative lifespans without malignant transformation or loss of the cell functions [11]. However, there are some limitations to this approach. For instance, the effectiveness of episomal TERT overexpression is constrained by only brief reconstitution of telomerase activity, [12] while the viral transfer of TERT induces random insertional mutagenesis to the genome and may compromise genetics and native behaviors of the primary cells. Moreover, exogenous TERT could evoke deleterious systematic immune responses in vivo owing to cellular immune responses against the exogenous TERT protein.

Over the past few years, CRISPR/dCas9-based epigenetic modifiers and transcriptional activators have been discovered as a biotechnological feat for site-specific manipulation of gene expression without the need to modify the underlying DNA sequence [13, 14]. Yet, to our knowledge, the use of CRISPR technology for extending the life of primary cells via activation of an endogenous TERT gene has not yet been reported so far. Importantly, the working principle of the CRISPR/dCas9-based tools implicates their applicability in telomerase-based immortalization of cells, including primary monocytes, circumventing the associated issues mentioned above. For instance, it has been reported that the epigenetic modifiers dCas9-p300 [14] and dCas9-TET1 [15] induce target gene activation by inducing H3K27ac histone acetylation and DNA demethylation, respectively, at the gene regulatory elements. They directly alter chromatin states or epigenetic marks on the promoter and enhancer regions to achieve stable target gene activation. On the other hand, the transcriptional activator dCas9-VPR is a VP64-p65-Rta tripartite activator; [16] and dCas9-VPH is a fusion of dCas9-VP192 and P65-HSF1 activator domains [17]. Both dCas9-VPH [17] and dCas9-VPR [16] are among the most potent programable transcriptional activators that can be used to briefly activate target endogenous gene expressions by recruiting multiple components of the transcription pre-initiation complex or histone acetyltransferases to the gene regulatory regions.

T cells represent around 60% of peripheral blood mononuclear cells (PBMCs) and are in a quiescence state when unstimulated [18]. A T cell rapidly divides upon stimulation, but it undergoes replicative senescence and will stop dividing when the telomere length is critically shortened through a finite number of cell divisions [19]. Studies have shown that the telomerase expression and activity were strongly correlated with the degree of stimulation-induced proliferation and survival of T cells in vitro [20]. T cells can upregulate telomerase activity to compensate for progressive telomere loss incurred during cell division in response to antigenic engagement of the T-cell antigen receptor (TCR) and co-stimulatory receptors, or exposure to specific cytokines, growth factors, and monoclonal antibodies [21–23]. However, telomerase reactivation through these stimulation approaches is brief and cannot prevent the ultimate onset of senescence due to the continued shortening of telomeres during in vitro long-term culture. Moreover, small-molecule-based methods can trigger cellular immune responses and globally alter the epigenome and transcriptome, thereby compromising the native function of T cells.

Here, we envision extending the replicative lifespan of T cells derived from primary PBMCs by activating its endogenous TERT expression using CRISPR/dCas9-based epigenetic modifiers (dCas9-p300 [14] and dCas9-TET1 [15]) and transcriptional activators (dCas9-VPH [17] and dCas9-VPR [16]). We further investigate that the combination use of these epigenetic modifiers and transcriptional activators can provide more robust, long-lasting endogenous TERT expressions in the primary cells. In principle, these programmable CRISPR tools can immortalize a variety of human/mammalian cells and overcome some of the limitations associated with exogenous telomerase-related methods. Ectopic telomerase overexpression or knock-in with telomerase gene was sufficient to elongate telomere lengths, sustain proliferation, increase population doublings, and extend replicative lifespans of primary T cells [11, 24]. Moreover, immortalization of human T lymphocytes has also been achieved by ectopic expression of the hTERT gene [25]. For validation, we also evaluate the effectiveness of these CRISPR tools in upregulating the endogenous TERT gene on other cell lines, including human embryonic kidney cells HEK293FT and human macrophages derived from monocytic leukemia cells.

Results

Targeted activation of endogenous TERT promoter by multiple single guide RNAs

CRISPR/dCas9-based epigenetic modifiers (dCas9-p300 [14] and dCas9-TET1 [15]) and transcriptional activators (dCas9-VPH [17] and dCas9-VPR [16]) were used to activate the endogenous human TERT gene by targeting the TERT promoter. To enable synergistic activation, three sgRNAs were designed to target the TERT promoter (Fig. 1a). These sgRNAs are − 157 bp, − 333 bp, and − 499 bp from the transcription start site (TSS) of the TERT gene. Since the human U6 promoter was used to drive the sgRNA expression, all our sgRNAs bear “G” at the 5’ end of the sgRNA sequence to initiate transcription. The 20 bp target sequence is immediately followed by a 3-bp 5’-NGG-3’ PAM sequence. Importantly, both CRISPOR (the track “CRISPR Targets” of UCSC Genome Browser) and Cas-Designer (the track “Cas-OFFinder”) showed all the designed sgRNA target sequences have a high targeting specificity across the human genome.

Fig. 1 CRISPR constructs for targeted gene activation. (a) Targeted activation of endogenous human TERT by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators. Red arrows indicate the sense or antisense orientation of sgRNAs (1, 2, and 3) designed to recognize target DNA sequences on the promoter. Plasmid constructs of CRISPR/dCas9-based epigenetic modifiers (dCas9-p300 and dCas9-TET1) and transcriptional activators (dCas9-VPH and dCas9-VPR) are also shown. (b) GFP-transfected cells. Plasmid CMV-GFPs were introduced to the PBMCs and THP-1-derived macrophages by nucleofection, while lipofectamine 3000 was used to transfect plasmids into the HEK293FT cells. The GFP fluorescence microscopic images were taken two days after plasmid transfections. Scale bar: 100 μm. (c) Results of the flow cytometry measurement of GFP signals

Transfection efficiency of PBMCs, THP1-derived macrophages, and HEK293FT cells

Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells containing CD3 + T cells, B cells, NK cells, monocytes, and dendritic cells [26]. The ratios of these cell populations vary across individuals. As the widely used liposome-based transfection reagents such as Lipofectamine 3000 can hardly transfect human leukocytes, nucleofection has been used to introduce the plasmids into PBMCs, with the optimized settings. To determine such optimized nucleofection, we have considered the macrophages derived from a human monocytic cell line (THP-1) with different experimental parameters. For instance, nucleofection of 1 × 106 cells in 20 µl of the reagents required a dose of 1000ng vector for the highest transfection efficiency. Notably, though hTERT expression in THP-1 cells is well-reported [27], we have quantified that THP-1-derived macrophages have a very low hTERT expression. Therefore, it is interesting to examine whether our CRISPR/dCas9-based approach can transfect THP-1-derived macrophages with induced expressions of TERT mRNA and the corresponding hTERT protein. To examine the transfection efficiency, we transfected the pCMV-GFP plasmids into the THP-1-derived macrophages and PBMCs by the optimized nucleofection transfection procedures and counted the cells with/without the GFP expression (Fig. 1b). We show that 15.46% and 19.54% of cells can be successfully transfected for THP-1-derived macrophages and PBMCs, respectively.

On the other hand, HEK293FT is a highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen [28, 29]. HEK293 was originally immortalized by adenoviral DNA (including the E1A and E1B genes) inserted on chromosome 19 that inhibited apoptosis and interfered with the transcription and cell cycle control pathway. Long-term cultivation and subcloning of HEK293 should result in genomic instability and karyotypic drift due to chromosomal translocations and copy number alterations. HEK293 exhibits tumor-like characteristics but they are not malignant yet. We transfected the pCMV-GFP plasmids into HEK293FT cells using Lipofectamine 3000 with an efficiency of 69.35% (Fig. 1c).

Targeted upregulation of endogenous TERT mRNAs and proteins

We transfected the selected cell types using the same procedures as described above for GFP and the CRISPR plasmids (See “Materials and Methods”). We considered two plasmid mixtures: (1) the epigenetic modifiers (dCas9-300 and dCas9-TET1) and (2) both the epigenetic modifiers and the transcriptional activators (dCas-VPH and dCas-VPR). To verify the effectiveness of our designed sgRNAs and CRISPR tools, we applied qPCR to quantify the hTERT mRNA, Western blot, and flow cytometry to measure the protein expressions. Our result (Fig. 2) shows that co-transfection with the epigenetic modifiers can upregulate the hTERT mRNA expressions of PBMCs (2.65 times) (Fig. 2a), THP-1-derived macrophages (53 times) (Fig. 2b), and HEK293FT cells (1.87 times) (Fig.2c). The minor upregulation for HEK293FT cells (Fig. 2c) can be explained by the fact that they may show some baseline expression of telomerase such that it is harder to further activate their hTERT promoter and upregulate their telomerase expression. Additionally, our result shows that co-transfection with both the epigenetic modifiers and the transcriptional activators induces significant upregulation of hTERT mRNA in all the selected cell types: PBMCs (2.85 times) (Fig. 2a), THP-1-derived macrophages (42.96 times) (Fig.2b) and HEK293FT cells (4.21 times) (Fig. 2c).

Fig. 2 TERT mRNA expression levels. Targeted activation of endogenous TERT mRNA in (a) PBMCs, (b) THP-1-derived macrophages, and (c) HEK293FT cells. qPCR was carried out after three days of plasmid transfection. Cells transfected with a single activator with or without sgRNA take part in, transfected with both epigenetic effector with SgRNA and a combination of epigenetic and transcriptional activators with sgRNA, a total of 11 groups of each cell line. The mRNA expression levels are scaled relative to the case for untreated cells. qPCR was carried out after three days of plasmid transfection. The statistical significance levels from untreated cells are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. All data are presented as mean ± SD (n = 3)

To evaluate the potency of each CRISPR tool in TERT reactivation, we transfected HEK293FT and PBMC cells with individual CRISPR tools (one type of activator) with or without the three chosen sgRNAs as mentioned in Fig. 1a. Results (Fig. 2) reveal that the expression level of TERT mRNA can only rise when sgRNA is involved. Briefly, HEK293FT transfected with dCas-VPR/P300 alone was sufficient to upregulate TERT mRNA expressions (3.56 and 2.57 times, respectively). Penev at al. previously used CRISPRa to activate endogenous tert in fibroblasts proving this [12]. Moreover, Li et al. revealed proof supporting the effects of P300 on the TERT transcription process [30]. However, no upregulation of TERT mRNA was achieved in PBMC cells transfected with dCas-VPR and dCas-p300 alone. Moreover, for M0 macrophage, dCas-P300, TET1, and VPH also have the effect of increasing the expression of TERT mRNA. (15.76 times, 13.21, and 10.83 times compared with the control group) This illustrates that cell type affects activation. Therefore, we combined epigenetic modifiers (dCas9-300 and dCas9-TET1) and transcriptional activators (dCas-VPH and dCas-VPR) for more robust TERT reactivation in PBMC cells.

We quantified the TERT protein of the selected transfected cells by flow cytometer as shown in Fig. 3a-c. Expectedly, both the epigenetic modifiers and the transcriptional activators upregulated the TERT expression of PBMC, M0 macrophage, and HEK293FT cells. We further examined the expression of a T-cell marker CD3 for different cases of PBMCs two months after the transfection process. The presence of CD3 enriched upon telomerase reactivation in those treated PBMCs indicates the survival and, hence, successful transfection of the T cells. Collectively, these results demonstrate that the reported CRISPR/dCas9-based approach can effectively activate the endogenous TERT expression in the resting primary T cells.

Fig. 3 hTERT protein expression levels. (a-c) Targeted activation of endogenous TERT protein in PBMCs, THP-1-derived macrophages, and HEK293FT cells were measured four days after the plasmid transfection by flow cytometry. Fluorescein isothiocyanate (FITC)-labeled anti-TERT antibodies were applied to stain the cells before measurements. Additionally, PBMC indicates expression of a T-cell marker CD3 in PBMCs stained with the FITC-labeled anti-CD3 antibodies, measured two months after nucleofection by flow cytometry. The occurrence (count over the total cell number) for each case is scaled such that the maximum value equals 1. A threshold level (vertical hidden line) for each cell type is determined such that > 99% of the corresponding unstained and untreated cells fall below the threshold level of protein expression. The percentage of cells with protein expression beyond the threshold level is indicated in each plot. (d) Western blot analysis of TERT protein. Three days after cells were transfected with activator plasmids (dCas9-P300, dCas9-TET1, dCas9-VPR, dCas9-VPH), proteins were harvested for Western blot. GAPDH was used as a reference control. (e) Normalized protein expression levels. hTERT protein expression level was normalized to GAPDH. For HEK293FT, the lower TERT band in Fig. d was used for the quantification in 3e

We also utilized the Western blot to quantify the TERT expression of the concerned cells, with GADPH as a housekeeping gene, as shown in Fig. 3d and e. Normalized expressions of the hTERT protein are increased significantly in the transfected-PBMC groups compared to the control groups (2.3-fold for only epigenetic modifiers and 2.4 times increase for both epigenetic modifiers and transcriptional activators). A combination of epigenetic modifiers and transcriptional activators also can increase the TERT protein expression of M0 macrophages, by approximately 1.3 times. HEK293FT cells exhibit the same trend in terms of the induced TERT expressions upon different cell treatments, yet the statistical differences are smaller than those for the PBMC cases (Fig. 3e). This could be explained by the presence of a baseline expression of telomerase in HEK293FT, which is consistent with TERT mRNA expressions mentioned above (Fig. 2c).

Proliferation of the TERT-activated resting T cells

We cultured and monitored the cell density of PBMCs upon nucleofection for up to two months. The initial cell density was 1 × 106 cells/ml. The cells transfected with epigenetic modifiers and transcriptional activators exhibited a higher proliferation rate than the cells transfected with only epigenetic modifiers, implying a more robust endogenous TERT activation in the former case. We monitored the population expansion of PBMCs transfected with both the epigenetic modifiers and the transcriptional activators. We took brightfield microscopes to quantify the normalized cell count using image processing software (Image J, NIH) at different time points of the cell culture (Fig. 4a and b). The cell density of untreated and transfected PBMCs was quantified at different time points throughout an incubation period of two months. Our results showed telomerase reactivation was able to increase the proliferative rate of PBMCs and undergo additional population doublings, whereas the population of untreated decreased over time (Fig. 4b). However, such growth rates may not be representative of any specific cell type in PBMCs, which is a mixture of multiple cell types with each an individual growth rate. Furthermore, we stained and examined the distribution of cell size in the untreated PBMCs and the expanded and transfected PBMCs, measured by the forward scatter parameter (FSC) in flow cytometry as shown in Fig. 4c. The untreated cells contain three dominant size ranges. We expect that the largest group (L) belongs to monocytes, the medium-sized group (M) belongs to NK cells and some granulocytes, and the smallest group (S) belongs to lymphocytes and possibly a small portion of dendritic cells. Hence, this result suggests enrichment of the CD3 + T cells population upon the nucleofection of PBMCs, agreeing with the positive CD3 expression as mentioned above (Fig. 3a).

Fig. 4 Characterizations of our engineered T cells. (a) Brightfield micrographs of cells were taken after two months of nucleofection. PBMCs were transfected with a combination of dCas9-p300 and dCas9-TET1 or a combination of four different CRISPR plasmids (dCas9-p300, dCas9-TET1, dCas9-VPH, and dCas9-VPR). The initial cell density among treatment groups is the same (1 × 106 cells/ml). Scale bar: 20 μm. (b) Normalized cell count of the CRISPR-treated (dCas9-p300, dCas9-TET1, dCas9-VPH, and dCas9-VPR) PBMCs (left) and untreated PBMCs (right) as a function of time. Normalized cell count was measured by calculating the number of cells in a given area (0.62 × 0.82mm2). The number of cells in a given area is counted through microscope photography, followed by normalizing with the control group (day 3). (c) Forward scatter parameter (FSC) of the flow cytometry measurement for the untreated PBMCs before nucleofection (red) and the transfected PBMCs after cultured for two months (black). (d) PI-based cell cycle analysis of our CRISPR/dCas9-based immortalized T cells, Jurkat T cells, and PBMC cells. (e) Flow cytometry analysis of engineered-T cells, Jurkat-T cells, and PBMC cells upon labeling with CFDA-SE (0 h) and after culturing of these labeled cells for a culture period. Purple is an unstained cell; red is the cell that is analyzed immediately after being stained with CFDA-SE (0 h); Blue is the cell stained with CFDA-SE and cultured (144 h for transfected PBMCs and PBMCs, and 72 h for Jurkat cells.), pink is the engineered and untreated PBMCs maintained for 240 h, and green is the treated and untreated PBMCs cultured for 400 h. (f) Annexin V and PI-labeled cells in our engineered-T cell population that have been cultured for 3 months before cell viability was carried out. A positive-control case is provided on the left, prepared by cell culture with 10% DMSO for 12 h. (g) Karyotype analysis of T-cell after 2 months of plasmid transfections. PBMC has a stable karyotype after being transfected

Characterization of immortalized T cells

The cell cycle and proliferation rate of the CRISPR/dCas9-based immortalized T cells were further characterized and compared with those of Jurkat cells, which are a widely used immortalized human T-cell line for studying T cell leukemia, and with those of PBMCs where the T-cell was derived from. The PI stain was used to determine the four phases of the cell cycle, i.e., cell resting/enlargement (G1), DNA synthesis (S), preparation for cell division (G2), and splitting (M). Results reveal that the majority of the CRISPR/dCas9-based immortalized T cells and PBMCs were in the resting state (G1) (Fig. 4d), while the majority of Jurkat cells were in the DNA synthesis (S) and dividing states (G2/M), implying that the lifespan of the CRISPR/dCas9-based immortalized T cells was successfully extended without significantly accelerated cell division, which can be an indicator of tumorigenicity.

In addition, CFDA-SE was used to label the CRISPR/dCas9-based immortalized T cells and control cells, which were subsequently cultured for ten days. In principle, cell division of a cell results in redistributing half of its fluorescence in each of the two divided cells in the next generation, implying that the reduction of the fluorescence can reflect cell division. Figure 4e shows the fluorescence intensity of the transfected and untreated PBMCs before and after CFDA-SE staining for 240 h, and jurkat cells for 72 h. The CFDA-SE data showed that the proliferation rate of our CRISPRa-treated PBMCs (∼ 5.7 division over 240 h) is higher than the untreated PBMCs (∼ 4.9 division over 240 h). Multiple studies have shown that T cells in a resting state will proliferate only once every 3–4 days [31]. The growth of T cells should contribute to the overall population increments of the bulk PBMCs. Jurkat cells have ∼ 8.93 division over 72 h corresponding to the decrease in fluorescent signal. Further, we applied Annexin V staining for detecting apoptotic cells and PI staining for identifying dead cells in CRISPR/dCas9-based immortalized cells that have been treated with chemical compounds (e.g. 10% DMSO). Flow cytometry analysis showed that all DMSO-treated cells were Annexin-V FITC positive and PI positive, confirming the reliability of this assay in detecting cell viability and proliferation (Fig. 4f). However, no staining of Annexin V and PI was observed for CRISPRa-treated PBMCs after being maintained for two months, indicating the cells are viable. Figure 4g is the karyotype analysis of continuously cultured modified cells (transfected with 4 activators) for two months. The result shows that stable karyotype and no chromosomal structure abnormality were observed in the transfected cells, which can be additional proof of the safety of the reported modification method.

Occupancy of activators on TERT promoter

It was known that the activation of T lymphocytes was regulated by NF-kβ, c-Myc, and pRb pathway. Therefore, the transcriptional activation activity of these activators on the TERT promoter was examined with CHIP-qPCR. As dCas9-P300 was used as an activator in this study, the H3K27ac antibody was used to detect the P300 activity on the TERT promoter [32]. TET1 antibodies were used to detect TET1 activator [33], and c-Myc [34] and HSF1 [35] were used to track the VPR and VPH actions, respectively. The fold enrichment method was used to analyze the CHIP-qPCR data as shown in Fig. 5a. For HEK293FT, enrichments of H3K27ac, TET1, c-MYC, and HSF occupancy at the TERT promoter were observed in CRISPRa targeting the hTERT, but no enrichment in the control group in HEK293FT cells were observed. This implies a specific localization of these proteins to the TERT promoter. For PBMC cells, the effector occupancies of TET were observed after transfected with CRISPRa targeting the hTERT.

Fig. 5 ChIP-qPCR and telomerase activity assay. (a) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis of the DNA binding activity of transfected HEK293FT and PBMC cells. The qPCR results are used to quantify the enrichment of H3K27ac and TET1 at the TERT promoter. The target antibody is H3K27ac, TET1, c-MYC, and HSF. The negative control is rabbit IgG. All the data were normalized by the negative control. (b) Telomerase activity assay of HEK293FT, M0 macrophage, and PBMC cells. TSR8 is an oligonucleotide identical to the TS primer extended with eight telomeric repeats AG(GGTTAG)7 which can be used as a standard for quantitative analysis of TRAP products. The statistical significance levels from untreated cells are indicated as ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. All data are presented as mean ± SD (n = 3)

Telomerase activity

After we quantified the TERT protein, we proceeded to measure telomerase activity (Fig. 5b). The telomere repeat expansion assay (TRAP) approach was used to quantify the telomerase activity. We first plotted a standard curve based on TSR8 copies using qPCR. TSR8 is an oligonucleotide identical to the TS primer extended with eight telomeric repeats AG(GGTTAG)7 which can be used as a reference standard for quantitative analysis of TRAP products. The telomerase activity of PBMC cells transfected with the CRISPR/dCas9 activation effector group was noticeably increased. PBMC transfected with the four activators demonstrated 5.6 times increases, and the epigenetic modifiers group has a 3.4-fold increase. For HEK293 cells, it also has an upward trend, Cells transfected with P300 and TET1 have the highest telomerase activity, 1.32 times more, and another transfected group with 1.24 times greater.

Delayed cellular senescence

The senescent cell was characterized by the high expression of β-galactosidase enzymatic activity, as shown with blue dye staining (Fig. 6a). Our senescence assay showed that activator-transfected cells exhibited delayed aging, while the majority of the control cells transfected with GFP displayed cellular aging. Flow cytometry analysis revealed a reduction in β-galactosidase staining on CRISPRa-treated PBMCs (from 3.74 to 2.09%), macrophages (from 13.62 to 7.79%), and HEK293FT cells (from 25.24 to 4.94%), indicating telomerase reactivation can delay senescence in these cells albeit at low efficiency (Fig. 6b). Additionally, in support of the β-galactosidase staining assay, immunofluorescence analysis of the most well-established senescence marker p21 also confirmed telomerase reactivation delayed senescence in CRISPRa-treated PBMC, macrophage, and HEK293 cells (Fig. 7).

Fig. 6 Cell senescence assay with β-galactosidase staining. Aged cells were confirmed with (a) microscope imaging of HEK293 FT cells and (b) flow cytometry analysis of β-galactosidase staining cells (scale bar: 50 μm)

Fig. 7 Cell senescence assay with p21 senescent marker. Aged cells were confirmed with (a) immunofluorescent imaging staining (scale bar: 50 μm) and (b) Immunofluorescence analysis of the p21 senescent marker. The fluorescence microscopic images were taken two weeks after plasmid transfection. Cells were stained with a fluorophore-conjugated secondary antibody with a fluorescent counterstain (DAPI, 1ug, Thermo Fisher, D1306)

Discussion

Here, we have successfully delayed cellular senescence and extended replicative lifespans of unstimulated T cells derived from human PBMC by reactivating its endogenous TERT expression using a combination of CRISPR/dCas9-based epigenetic modifiers and transcriptional activators. Compared to exogenous telomerase-overexpressing methods, reactivation of endogenous telomerase can alleviate potential host immune response, such as the generation of neutralizing antibodies which may attenuate the cure effect of viral-mediated gene therapy. Thereby the reported method allows safer adoptive transfer of TERT-expressing T cells in vivo. Targeted reactivation of endogenous telomerase can also minimize the risk of developing genomic instability and malignant transformation in T cells, as it ensures a functioning physiological level of TERT expression. Moreover, viral-based transgenesis approaches may alter cell phenotypes due to long periods of drug selection for the desired genetically engineered cells. Although it has been reported that external stimuli (e.g., interleukin-2 [36], monoclonal antibodies CD3 and CD28 [37]) are capable of briefly reactivating endogenous telomerase gene in human T lymphocytes, such TERT reactivation is temporary and difficult to support subsequent stimulations for immunotherapy applications [11, 38].

On the contrary, the use of CRISPR/dCas9-based epigenetic modifiers in our study can prolong the TERT reactivation in the T cells by rewiring the epigenetic states that regulate TERT expression. However, TERT-overexpressing human T cells are unlikely to have malignant transformation. [39]; and their behaviors are long-lasting as the primary cells [40]. We have observed the TERT-expressing T cell lines for three months of culture after the epigenetic modification and verified that their actual replicative lifespans do not come with accelerated cell division in vitro. Most engineered T cells are in the G1 phase, whereas T lymphocyte cancer cells (Jurkat) are primarily in the S and G2 period. This data of cell cycle analysis together with the stable karyotype observed in engineered T cells, implicating the absence of malignant transformation.

As classical approaches for endogenous telomerase reactivation are rather inefficient and brief, TERT transgene can be inserted into the genome of the T cells using retrovirus or lentivirus vectors for prolonged hTERT expression. For example, the proliferative lifespan of T cells has been substantially extended through ectopic expressing TERT transgene [6, 7, 11]. Although these immortalized T cells have been continuously cultured for longer than one year without loss of primary cell functions and malignant transformation [6, 41, 42], this viral-based transgenesis approach may induce random insertional mutagenesis to the genome and may alter cell phenotypes due to long periods of drug selection for the desired genetically engineered cells [43]. On the contrary, CRISPR/dCas9-based epigenetic modifiers and transcriptional activators that we have used to activate the endogenous TERT promoter do not alter the DNA sequence at this genomic locus or induce any DNA damage to the genome. This method triggers physiological or near-physiological expression of the endogenous TERT gene by directly modulating its regulatory elements. In other words, it specifies downstream expressions of the most relevant splice variants from the TERT gene.

The reported CRISPR-based strategy can retain alternative splicing as in the unmodified cells, such that they can produce several forms of the TERT mRNA through post-transcriptional regulation [21]. It overcomes some key disadvantages of the ectopic overexpression approach, in which some of the physiologically relevant splice variants could be missed if their cloning expressions are not specified by the cDNA cassette [44]. Overexpressing a certain splice variant from the open reading frame (ORF) may also result in missing other physiologically relevant splice variants. In addition, protein overexpression can also be harmful to the cell since forced protein production can disrupt the balance between protein complexes and the underlying cell physiology.

In essence, this work exhibits that the transfected PBMCs contain immortalized T-cells, verified by CD3 expression, cell size distribution, as well as continuous cell proliferation. In addition to PBMC-derived T cells, we have demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. CRISPR/dCas9-based epigenetic modifiers and transcriptional activators could robustly upregulate the TERT expression in HEK293FT and THP-1-derived macrophages, despite no noticeable expression of hTERT at a baseline level. We envision that the strategy of extending the cell life described here can be applied and generalized to other primary human cells.

In addition, we have also attempted to combine different activators and determine which ones have effects on the TERT expression using chromatin immunoprecipitation (ChIP)-qPCR. ChIP has been demonstrated as a useful tool in the analysis of genome-wide effects of CRISPR-based transcription activators in human cells to access the specificity of DNA binding [45]. Our results illustrate that p300, TET1, VPR, and VPH are directly bound to the promoter of TERT to activate the TERT expression. Although VPR and VPH are very strong activators, their activation effects last only for several days. In fact, our previous studies have shown that the mRNA and protein levels of the target gene were restored to the baseline level after 5 days if the VPR activator was used alone. Further, recent research illustrates enhanced combination effects of gene activation (p300 and VPR) compared to p300 or VPR alone, suggesting the cooperativity between transcription and epigenetic regulation [46]. p300 and TET1 epigenetic modifiers enable long-term target gene activation by directly altering chromatin states or epigenetic marks on the regulatory elements. Therefore, we combined the strong VPR and VPH activators with p300 and TET1 epigenetic modifiers to achieve robust, long-lasting endogenous TERT expressions of the gene-edited cells.

Materials and methods

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.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Abbreviations

ChIP Chromatin immunoprecipitation

CRISPR Clustered regularly interspaced short palindromic repeats

GFP Green fluorescent protein

HEK293 Human embryonic kidney 293

HSF1 Heat shock factor 1

hTERT Human telomerase reverse transcriptase

ORF Open reading frame

PBMC Peripheral blood mononuclear cell

PI Propidium iodide

TET1 Tet methylcytosine dioxygenase 1

THP-1 Human leukemia monocytic cell line

TRAP Telomere repeat expansion assay

VPH dCas9-VP192 and P65-HSF1 activator domains

VPR VP64-p65-Rta tripartite activator

Author contributions

L.R.H.W. and T.C. conceived and supervised the project. H.S.P. and L.C.H. performed the experiments, collected and analyzed the data. H.S.P., L.C.H., T.C., and L.R.H.W. wrote and revised the manuscript. All authors read, corrected, and approved the final manuscript.

Funding

This work was supported by Hong Kong Research Grant Council (General Research Grant 11215619).

Data availability

All the data and supporting materials are available within the article and Supplemental information.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Siping Huang and Cia-Hin Lau contributed equally to this work.
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