Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

CRISPR and HDR template design and assembly

CD226- and FOXP3-targeting gRNAs were synthesized as chemically modified crRNAs (IDT) and duplexed at a 1:1 molar ratio with tracrRNA (IDT) by denaturation for 5 min at 95°C followed by gradual cooling to room temperature. The resulting gRNA was complexed with Cas9 (QB3 Macrolab) at a 2:1 molar ratio for 10 min at room temperature. gRNA target sequences: CD226 (5′-GTTAAGAGGTCGATCTGACG-3′), FOXP3 CR1 (5′-AGGACCCGATGCCCAACCCC-3′), FOXP3 CR2 (5′-GGGCCGAGATCTTCGAGGCG-3′), FOXP3 CR3 (5′-GCAGCTGCGATGGTGGCATG-3′), FOXP3 CR4 (5′-TGCCCCCCAGCTCTCAACGG-3′), FOXP3 CR5 (5′-CCCACCCACAGGGATCAACG-3′). HDR designs contained homology arms of at least 800 bp flanking a promoterless or EF-1α-driven transgene marker (sfGFP or ΔNGFR). Fragments were PCR-amplified from genomic DNA, plasmids, or synthesized (IDT gBlocks or GenScript GenParts), then cloned by Gibson assembly into an AAV transfer plasmid between two ITRs.

AAV6 production, purification, and titration

For recombinant AAV6 production, HEK-293T/17 cells (ATCC CRL-11268) were co-transfected by calcium phosphate with an adenoviral pHelper plasmid, a pAAV6-Rep-Cap plasmid (both Cell Biolabs), and an ITR-flanked transfer plasmid at a 1:1:1 molar ratio. Cells were harvested after 3 d and AAV6 was purified with a filtration-based kit (Takara AAVpro Purification Kit) per the manufacturer’s protocol. AAV6 vector genomes were titrated by ITR-specific quantitative PCR (Takara AAVpro Titration Kit Ver.2) per the manufacturer’s protocol.

Cell isolation and culture

Human blood sample collection from healthy adults was performed in accordance with protocols approved by the University of British Columbia Clinical Research Ethics Board and Canadian Blood Services. CD3+ T cells or CD4+ T cells were isolated via Lymphoprep and RosetteSep (both STEMCELL Technologies). For Treg isolation, CD4+ T cells were enriched with CD25 MicroBeads II (Miltenyi Biotec) before flow sorting on a MoFlow Astrios (Beckman Coulter) or FACSAria IIu (BD Biosciences). Sorting strategies: total Tregs (CD4+CD25hiCD127lo), naive Tregs (nTreg; CD4+CD25hiCD45RA+CD127lo), memory Tregs (mTreg; CD4+CD25hiCD45RA−CD127lo), and conventional T cells (Tconv; CD4+CD25loCD127hi). Unless otherwise indicated, all cells were cultured at 37°C, 5% CO2 in X-VIVO 15 (Lonza) supplemented with 5% (v/v) human serum (WISENT), 1% (v/v) penicillin-streptomycin (Gibco), 2 mM GlutaMAX (Gibco), and 15.97 mg/L phenol red (Sigma-Aldrich). During optimization, in some cases as indicated, CTS OpTmizer T Cell Expansion SFM (Gibco) was used in place of X-VIVO 15, human serum, and phenol red.

Human Treg expansion with CRISPR/HDR-mediated gene knock-in

In all cases during pre-activation and expansion, media was supplemented with IL-2 (Proleukin; 1000 IU/ml for Tregs, 100 IU/ml for Tconvs or CD4+ T cells). Tregs, Treg subsets, Tconvs, and CD4+ T cells were pre-activated with either CD3/CD28/CD2 tetrameric antibody complexes (ImmunoCult by STEMCELL Technologies) or 1:1 aAPCs for 5 d. aAPCs were L cells (ATCC CRL-2648) expressing CD32, CD58, and CD80, gamma-irradiated (75 Gy), and loaded with and anti-CD3 (OKT3, 0.1 μg/ml; University of British Columbia Antibody Lab) (de Waal Malefyt et al., 1993). Media and IL-2 were replenished every 2–3 d.

Cells were electroporated with the Neon Transfection 10 μL Kit (Invitrogen) per the manufacturer’s protocol. Briefly, cells were washed twice with PBS, resuspended in Buffer T (≤20×106 cells/mL) with Cas9 or RNP (40 pmol gRNA + 20 pmol Cas9 per transfection), electroporated at 1400 V / 30 ms / 1 pulse, then immediately transferred into prewarmed antibiotic-free media containing AAV6 at the specified vg/cell and simultaneously expanded with aAPCs and IL-2 for 7 d as above. After 7 d expansion (total 12 d), cells were flow-sorted as sfGFP+, then rested overnight in reduced IL-2 (100 IU/ml for Tregs, none for Tconvs) before functional assays; in some cases, flow-sorted cells were further expanded for 7 d with aAPCs and IL-2 as above before use in assays. Cell counts at 5 d were determined by trypan blue (Gibco); cell counts at 12 d and 19 d were determined by ViaStain Acridine Orange/Propdium Iodide Staining Solution (Nexcelom).

For experiments investigating the role of FOXP3 in Treg biology, we pre-activated cells with CD3/CD28/CD2 tetramers followed by aAPC-based expansions, for two reasons. First, Treg yield by day 19 of expansion was comparable to a fully aAPC-based expansion (Figure 1D). Second, we found that repetitive (3×) aAPC-based stimulation occasionally resulted in spontaneous FOXP3 loss (data not shown), which could be circumvented by using a weaker reagent to pre-activate Tregs, i.e., CD3/CD28/CD2 tetramers.

During optimization, cells were alternatively pre-activated with IL-2 as above and one of: 1:1 anti-CD3/anti-CD28-coated beads (Gibco Dynabeads Human T-Expander or Human Treg Expander), CD3/CD28 tetrameric antibody complexes (ImmunoCult by STEMCELL Technologies), or a combination of plate-bound anti-CD3 (OKT3, 10 μg/ml in 0.1 M Trizma HCl; Sigma-Aldrich) with soluble anti-CD28 (CD28.2, 4 μg/ml; BD Biosciences). Dynabead-activated cells were magnetically de-beaded immediately prior to electroporation. Divergent T cell pre-activation conditions and Neon electroporation parameters are specified as applicable. In some cases, HDR Enhancer (IDT, v1) was added to AAV6-containing media prior to electroporation at the indicated concentrations.

Evaluation of genome editing

To assess indel formation after CRISPR editing, genomic DNA was extracted from cells 3 d after electroporation using a QIAamp DNA Mini Kit or Micro Kit (QIAGEN) per the manufacturer’s protocols. The region flanking the gRNA cut site was PCR-amplified, Sanger-sequenced, and chromatograms uploaded to the ICE analysis tool (Synthego; (Hsiau et al., 2019). Primer sequences for FOXP3 CR1 and FOXP3 CR3: FWD (5′-CTAGAGCTGGGGTGCAACTATG-3′), REV (5′-TCTTCTCTTGTCACATGGGGATG-3′). Primer sequences for FOXP3 CR5: FWD (5′-GAATGGCCGTCTTTAAGCTTCTC-3′), REV (5′-TTATTGGGATGAAGCCTGAGCTG-3′). An asterisk (*) denotes the primer used for Sanger sequencing.

T cell suppression assay

Expanded Tregs and ex vivo-isolated allogeneic CD3+ T cells (responder cells) were labelled with Cell Proliferation Dye (Invitrogen) eFluor 670 and eFluor 450, respectively, then cocultured at the indicated ratios and activated with anti-CD3/anti-CD28-coated beads (Gibco Dynabeads T-Expander; 1:16 bead:responder cells) for 4 d. Proliferation of CD4+ and CD8+ cells within the responder-cell fraction was determined by dilution of Cell Proliferation Dye; responder cells activated with beads served as positive controls. Percent suppression of proliferation was calculated as: (1 − (division index of sample / division index of positive control)) * 100.

Assessment of cytokine production

Expanded Tregs or Tconvs were activated with IL-2 (100 IU/mL) and 1:1 anti-CD3/anti-CD28-coated beads (Gibco Dynabeads T-Expander) for 4 d. For secreted cytokines, supernatants were collected, cytokines measured by cytometric bead array (BioLegend LEGENDplex Human Th Panel 13-plex), and data analysed with Qognit software (BioLegend) per the manufacturer’s protocols. For intracellular cytokine expression, cells were restimulated with PMA (10 ng/ml), ionomycin (500 μg/ml), and brefeldin A (10 μg/ml; all Sigma-Aldrich) for an additional 4 h.

Flow cytometry

Antibodies are listed in Table S2. Cells were stained for surface proteins in PBS (Gibco) or Brilliant Stain Buffer (BD Biosciences) for 20 min at room temperature; Fixable Viability Dye (Invitrogen) or 7-AAD (BioLegend) was used to exclude dead cells. For detection of intracellular proteins, cells were fixed and permeabilized with the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) for 40 min at room temperature, then stained for intracellular proteins for 40 min at room temperature. As applicable, Brilliant Stain Buffer Plus (BD Biosciences) was included during intracellular staining. Samples were acquired on an LSRFortessa X-20, FACSymphony A5 (both BD Biosciences), or Cytoflex (Beckman Coulter), and data were analysed with FlowJo software (BD Biosciences; v10.7). All live single cells were first gated as CD4+, except for the T cell suppression assay in which live single cells were gated as CD4+ or CD8+. Percent protein KO was calculated as: (1 – (% marker+ of RNP-edited sample / % marker+ of negative control)) * 100.

Genome-wide DNA methylation profiling

FOXP3HDR-KO nTregs, Cas9 nTregs, and Tconvs expanded for 20 d from 5 individuals were used for genome-wide DNA methylation profiling. Genomic DNA was isolated with an AllPrep DNA/RNA Mini Kit (QIAGEN), bisulfite-converted with an EZ DNA Methylation Kit (Zymo Research), hybridized to Infinium MethylationEPIC BeadChips (Illumina), and imaged using an iScan System (Illumina) per the manufacturers’ protocols. Raw red and green intensity data (IDAT) were imported into the R statistical environment (v4.0.2) with RStudio (v1.3.1093), converted into methylated and unmethylated signals, normalized using noob (Triche et al., 2013) and functional normalization (Fortin et al., 2014) by preprocessFunnorm in minfi (v1.34.0) and by BMIQ (Teschendorff et al., 2013) in wateRmelon (v1.32.0), and corrected for batch effects (BeadChip ID and position) using ComBat in sva (v3.36.0). Probes with a detection p-value > 0.01 or a bead count < 3 in at least one sample were discarded. Probes previously characterized to have low-quality genomic mapping, cross-reactive or polymorphic sequences, or type I probes targeting a common SNP that could cause a colour channel switch were discarded (Zhou, Laird and Shen, 2017) ( Because the cohort contained cells from males and females, probes targeting X/Y chromosomes were discarded. The total remaining CpG sites after preprocessing was 702,481. Results were visualized as beta values, which range from 0 (unmethylated) to 1 (fully methylated).

Principal component analysis was performed using prcomp. Differential methylation analysis for all paired comparisons was performed with logit-transformed beta values (M values), considered more statistically robust (Du et al., 2010), using limma (v3.44.3). CpG sites with a mean methylation (beta) difference > 0.05 and Benjamini-Hochberg-corrected p-value < 0.05 were considered differentially methylated. DMRs were then identified by DMRcate (v2.2.3) (Peters et al., 2015) using a bandwidth of 1000 nt (lambda = 1000) and a scaling factor of 2 (C = 2). Regions with contiguous, differentially methylated CpG sites within lambda nucleotides, a region mean methylation (beta) difference > 0.05, and Fisher’s multiple-comparison statistic < 0.05 were considered significantly differentially methylated.

To identify differentially methylated regions that overlapped a FOXP3-binding region, a publicly available human FOXP3 whole-genome ChIP-chip tiled array dataset was used (Sadlon et al., 2010). The genomic coordinates of FOXP3-binding sites identified by model-based analysis of tiling-arrays were lifted over from hg18 to hg19, then compared to the DMRs identified above. A DMR was considered overlapping a FOXP3-binding region if there was an overlap of at least 1 nt.

HOMER (Heinz et al., 2010) was used for transcription factor binding motif enrichment analysis. The coordinates of demethylated DMRs were scanned using with default parameters, exact (given) fragment size, and “-mask” to mask repeat sequences. Background sequences with matching GC content were randomly selected as controls. Demethylated DMRs were searched for overrepresented motifs with lengths of 8, 10, and 12 bp relative to the background sequences. De novo motifs with p-value < 1×10−12 were considered enriched.

Statistical analysis

Normality was assumed. Statistical significance between more than two groups was determined by matched 1-way ANOVA or matched 2-way mixed-effects model and Dunnett’s or Tukey’s multiple comparisons tests, as appropriate. Significance between two groups was determined by paired t-test; for lognormal distributions, ratio paired t-test or Mann-Whitney test was used, as appropriate. For AAV6 dose response experiments, areas under the curve were used to determine significance. For suppression of proliferation, areas under the curve were used to determine significance, taking into account all degrees of freedom, as previously described (Akimova et al., 2016). The n values used to calculate statistics are defined in the figure legends; significance (p < 0.05 was considered significant) is indicated within the figures. Analysis was performed using Prism software (GraphPad; v9.0.0). Statistical testing of DNA methylation patterns was performed in R, as described in the preceding section.

Article TitleOptimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity


Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

Competing Interest Statement

MKL received research funding from Sangamo Therapeutics, Bristol-Myers Squibb, Pfizer, Takeda, and CRISPR Therapeutics for work unrelated to this study. All other authors declare no competing interests.

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