Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Summary

This protocol describes a non-integrative, episomal CRISPR/dCas9-based system for targeted epigenetic editing in K562 cells, combining dCas9-DNMT3A and dCas9-HDAC1 effectors with specific sgRNAs to induce locus-specific DNA methylation with precision and reduced off-target effects.

Abstract

Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.

Introduction

Epigenetic regulation, including DNA methylation and histone modifications, plays a pivotal role in controlling gene expression, cell differentiation, and genome stability. Dysregulation of these mechanisms is often involved - either as a cause or a consequence - in various pathological states, particularly in cancer, neurological disorders, and imprinting syndromes^1,2,3. Notably, by precisely intervening to the epigenetic landscape at specific genomic loci using epigenome editing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, gene repression levels comparable to those observed in gene knock-out models can be achieved, without altering the underlying DNA sequence⁴. As a result, targeted epigenetic editing has emerged as a promising strategy for dissecting gene regulatory mechanisms and developing precision therapeutics.

DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are epigenetic therapies currently approved for clinical use. However, these agents non-selectively target epigenetic markers across the genome, altering global chromatin states, causing off-target effects and toxicity in patients⁵. To address the limitations of traditional epigenetic alteration agents, programmable epigenetic editors based on catalytically dead Cas9 (dCas9) fused to effector domains, such as DNA methyltransferases (e.g., DNMT3A), demethylases (e.g., TET1), histone acetyltransferases (e.g., p300), or deacetylases (e.g., HDAC3), have been developed^6,7,8,9. The dCas9-effector system allows for locus-specific epigenome modification guided by a small RNA molecule, known as the single-guide RNA (sgRNA), without introducing double-stranded breaks at the target sequence. Recent studies have demonstrated their use in modifying gene expression, remodeling chromatin accessibility, and even reprogramming cellular phenotypes^10,11,12.

Despite rapid advances, dCas9-based epigenome engineering systems continue to face some limitations. The most common challenge is off-target effects due to the nonspecificity of epigenetic effector domains, especially with strong or prolonged expression of the dCas9 fusion tools^13,14. Additionally, the large size of dCas9 fusions presents significant delivery challenges. The size of the Streptococcus pyogenes Cas9 (SpCas9) sequence is approximately 4.2 kb¹⁵, which is comparable to the maximum capacity size of Adeno-associated virus (AAV) vectors. While lentiviral vectors have a relatively large capacity (~12-1 kb), enabling delivery of the full-length SpCas9 fused with epigenetic effectors, they still retain the possibility of random genomic integration, risking insertional mutagenesis and limiting their clinical applications¹⁶. On the other hand, non-viral approaches such as plasmid transfection suffer from poor retention in dividing cells. Transient or "hit-and-run" delivery approaches exclude the possibility of genomic integration, but often fail to sustain methylation changes across cell divisions, unless combined with chromatin remodelers or cooperative effectors^17,18,19.

To address these challenges, we employed a non-integrative, self-replicating episomal vector system (pEPI-S/MAR) for delivering dCas9-fused epigenetic effectors. This vector persists extrachromosomally in dividing cells at low copy numbers (~1-2 per cell), allowing for sustained yet moderate expression, in order to minimize undesired functions associated with overexpression^20,21. The Scaffold/Matrix Attachment Region (S/MAR) element of the episome interacts with the nuclear matrix, contributing to the episome's stable maintenance in the cells' nucleus during cell division^22,23. Furthermore, the pEPI vector system avoids risks associated with viral integration and enables delivery of constructs without exceeding packaging limits.

Recent studies have shown that promoter regions marked by active histone modifications, particularly H3K27 acetylation (H3K27ac), are often resistant to engineered DNA methylation. Combinatorial approaches that either remove or add histone marks using histone modifications' writers or erasers and simultaneously introduce DNA methylation via DNMT3A have been shown to increase the stability and persistence of gene repression, even in transient approaches^19,24. Based on these insights, we hypothesized that co-delivering dCas9-HDAC1 and dCas9-DNMT3A and targeting neighboring genetic areas would promote chromatin remodeling and facilitate more efficient and durable methylation at target regulatory elements.

In this study, we aimed to design and construct a system capable of delivering targeted and stable DNA methylation to CpG island 326 of ZBTB7A, a region characterized by low endogenous methylation and aberrant H3K27ac marks in K562 cells. The objective of this study was to deliver two episomal constructs encoding dCas9 fused to each of the distinct epigenetic effectors, along with sgRNAs, targeting the CpG-rich regulatory region upstream of the ZBTB7A gene using the low-expression, stable, non-integrative pEPI system. As illustrated in Figure 1, co-transfection of K562 cells with these episomes enables the dual recruitment of the editing complexes to the CpG island of interest, where HDAC1 removes local histone acetylation to facilitate subsequent DNA methylation by DNMT3A, resulting in coordinated and site-specific epigenetic modification.

The system described here is appropriate for applications where viral delivery is not feasible due to construct size, safety concerns, or cost limitations. It supports modular cloning of different sgRNAs and effector fusions, making it adaptable to a range of target loci. It is currently best suited to in vitro or preclinical studies in cell lines that tolerate plasmid transfection and antibiotic selection. In our implementation, episomal delivery in K562 cells was achieved using lipofectamine-based reagents and maintained under G-418 selection.

Protocol

1. Design of sgRNAs

1. Identify the desired CpG island or regulatory CpG sites within the promoter region of the gene of interest using a genome browser (e.g., UCSC genome browser). Here, the design was applied on the CpG island 326 (GRCh38)of the ZBTB7A gene.
2. Design single guide RNAs (sgRNAs) using an online CRISPR sgRNA design platform [e.g., CHOPCHOP web tool (version 3)]. When designing sgRNAs, follow the recommendations below to ensure high on-target activity and minimal off-target effects:
   1. Select SpCas9 protein as the endonuclease, using 5'-NGG-3' as the protospacer adjacent motif at the 3' end (PAM-3').
   2. Choose sgRNA length to be 20 bp (besides the PAM sequence). Allow off-target matches with only three mismatches in the protospacer sequence.
   3. When selecting to target a specific region of the gene, choose the 'Promoter' option and define the upstream range (e.g., from 1500 bp to 3000 bp upstream for CpG island 326) to position the sgRNAs within the regulatory region.
   4. Use on-target efficiency scores such as those developed by Doench et al.²⁵, to assess sgRNA activity.
3. When selecting sgRNAs for experimental use, consider the following criteria:
   1. Rank all candidate sgRNAs by efficiency for the target region. Choose sgRNAs in the top 10-20% of predicted efficiency. When choosing, avoid sgRNAs with mismatches (MM0, MM1, MM2, MM3) at potential off-target sites.
   2. Choose sgRNAs that target originally unmethylated or weakly methylated areas for transcriptional repression using dCas9-DNMTs. For transcriptional repression using dCas9- HDACs, target highly acetylated regions for increased efficiency. Use online databases (e.g., ChIP-Atlas) to confirm DNA methylation and histone acetylation marks in K562 cells at the target loci.
   3. Use multiple sgRNAs to cover target regions larger than 100-200 bp²⁶.
      NOTE: Epigenetic modifications (methylation and deacetylation) influence regions upstream and downstream of the sgRNA binding site, and these effects might be limited to short genomic spans.
   4. Choose sgRNAs with a GC content between 40% and 80%.
      NOTE: The GC content of the sgRNA is important, as higher GC content improves RNA stability and dCas binding.
   5. Avoid sgRNAs containing stretches of four or more thymidines (Ts) and choose sgRNAs with adenine or thymine nucleotides located four bases upstream of the PAM site.
      NOTE: Four or more Ts can lead to premature transcription termination. sgRNAs with A or T located four bases upstream of the PAM site may exhibit stronger dCas binding.
   6. Check and avoid sgRNAs with high self-complementarity to minimize the risk of secondary structure formation.
   7. Finalize the selection of a few sgRNA sequences.
      NOTE: Four sgRNAs were designed spanning across the CpG island 326. The sgRNA1-4 target sequences and target positions across CpG island 326 are shown in Supplementary Table 1.
4. Transform the sgRNA sequence of choice (excluding the PAM triplet) into its reverse complement. Add GATCG to the 5' end of the forward sequence and G to its 3' end, and AAAAC to the 5' end of the reverse complementary sequence and C to its 3' end. Order the two final oligos for each sgRNA from a commercial oligo provider.

2. Cloning

NOTE: This section describes the tailoring of plasmid pEPI-1 to generate episomal vectors carrying both the dCas-epigenetic effector fusion and sgRNA expression cassettes. See Figure 2 for a comprehensive overview of the cloning workflow and vector architecture. All restriction digestion and ligation reactions described below were carried out according to the manufacturer's recommended protocols for each enzyme and reagent used, and reaction specifications are listed in detail in Supplementary Table 2.

1. Digest the pEPI-1 plasmid using AgeI and BspEI, which flank the eGFP (enhanced green fluorescent protein) coding sequence. Following restriction digestion, blunt the resulting overhangs using the Klenow fragment of DNA polymerase I. Ligate the blunt-ended plasmid backbone using T4 DNA ligase to generate the eGFP-deleted vector.
   NOTE: Deletion of the eGFP gene is performed to reduce the overall vector size. A larger vector size might limit the downstream cloning and transfection steps.
2. Obtain the coding sequences for the catalytic domain (CD) of the DNMT3A gene and the full-length of the HDAC1 gene. Append a stop codon at the 3' (downstream) end of each coding sequence. Add restriction sites BglII (5') and AseI (3') flanking the sequences.
3. Digest the eGFP-deleted pEPI-1 vector with BglII and AseI. Separately, digest the prepared DNMT3A(CD) and HDAC1 coding sequences with the same enzymes. Perform two independent ligation reactions:
   1. Ligate the DNMT3A(CD) fragment into the digested vector to generate the pEPI-1-DNMT3A construct.
   2. Ligate the HDAC1 fragment into the digested vector to generate the pEPI-1-HDAC1 construct.
      NOTE: BglII and AseI sites place the inserts downstream of the pCMV promoter and upstream of the S/MAR element for optimal expression.
4. Transform chemically competent Escherichia coli (e.g., NEB 5-alpha Competent E. coli, High Efficiency) with the ligation products from each vector separately. Plate the transformed cells on LB agar containing kanamycin (50 µg/mL) and incubate overnight at 37 °C. Select positive colonies and isolate plasmid DNA using the rapid boiling miniprep method^27,28.
5. Obtain the sequence encoding the catalytically dead SpCas9 (dCas9). Introduce the following elements in the specified order:
   1. At the 5' end: AscI restriction site, Kozak consensus sequence, and start codon (ATG).
   2. At the 3' end: NLS, optionally an HA-tag, followed by a BamHI restriction site.
6. Digest both pEPI-1-DNMT3A and pEPI-1-HDAC1 vectors with AscI and BamHI. Perform the same digestion on the dCas9 insert. Ligate the dCas9 fragment into each respective vector.
   NOTE: The AscI/BamHI sites ensure that dCas9 is inserted upstream of DNMT3A or HDAC1, directly downstream of the pCMV promoter. Ensure that a short linker or a few nucleotides are present between dCas9 and the epigenetic effector to maintain the correct reading frame.
7. Transform the final ligation products into high-efficiency competent E. coli strains with tight control of plasmid expression (e.g., NEB 5-alpha F'Iq Competent E. coli, High Efficiency). Select transformants on kanamycin plates. Prepare high-quality plasmid DNA from positive clones using the rapid boiling miniprep method.
8. Sequence the final plasmid constructs (pEPI-1-dCas9-DNMT3A and pEPI-1-dCas9-HDAC1) using nanopore sequencing to confirm the correct assembly of all components, verify the reading frame using an online translation tool (e.g., Expasy translate tool 3.0), and check for potential mutations or recombination events using an online alignment tool (e.g., BLAST alignment tool).
   NOTE: Large vectors are more prone to recombination and may accumulate mutations. Therefore, thorough sequence validation is essential.
9. Phosphorylate the 5' hydroxyl groups of both forward and reverse sgRNA oligonucleotides using T4 polynucleotide kinase. Hybridize the two complementary strands by heating the mixture to 90 °C for several minutes to denature secondary structures, then allow gradual cooling to room temperature overnight for complete annealing into double-stranded DNA.
10. Digest the selected pGuide sgRNA expression vector with BamHI and Esp3I. Ligate the annealed double-stranded sgRNA fragment into the linearized vector using T4 DNA ligase.
11. Using specific PCR primers (Supplementary Table 3), amplify the full gRNA expression cassette comprising (1) U6 promoter, (2) Cloned sgRNA sequence, and (3) sgRNA scaffold. Use a high-fidelity DNA polymerase that does not generate 3' overhangs (i.e., a proofreading polymerase) to ensure clean, blunt-ended PCR products suitable for downstream cloning.
12. Repeat steps 2.9-2.11 for each individual gRNA designed in section 1. Ensure each gRNA cassette is prepared and amplified separately to preserve target specificity.
13. Digest the vectors pEPI-1-dCas9-DNMT3A and pEPI-1-dCas9-HDAC1 with SacII. Blunt the sticky ends using the Klenow fragment of DNA polymerase I. Clone the previously amplified U6 promoter-sgRNA-scaffold cassettes into these vectors using T4 ligase.
    NOTE: Several gRNA-epigenetic effector combinations (e.g., sgRNA1-dCas9-DNMT3A, sgRNA2-dCas9-HDAC1, etc.) were constructed during the experimental phase. However, only two selected combinations were carried forward for the experiments presented in subsequent sections.
14. Transform the final ligation products into high-efficiency competent E. coli strains with tight control of plasmid expression. Select transformants on kanamycin plates. Prepare high-quality plasmid DNA from positive clones using the rapid boiling miniprep method.
15. Sequence the final plasmid constructs using nanopore sequencing, verify the reading frame, and check for potential mutations or recombination events.

3. Cell culture and transfection

1. Maintain K562 cells in T-75 culture flasks at 37 °C, under 5% CO₂ and humidity conditions. Use Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1× Penicillin/Streptomycin solution (Pen/Strep).
2. Prepare plasmids for transfection:
   1. Use 8 µg of total plasmid DNA per transfection, hence 4 µg of each plasmid for a co-transfection of two plasmids. Quantify the DNA using absorbance at 260 nm, and then dilute the required amount in 90 µL of ultra-pure water.
   2. Add 10 µL of 3 M Sodium Acetate solution and 2.5 volumes of 100% ethanol to the diluted DNA.
   3. Mix thoroughly and incubate at -20 °C overnight to precipitate the DNA.
      NOTE: When co-transfecting two plasmids with distinct gRNA-epigenetic effector combinations (e.g., gRNA1-dCas9-DNMT3A and gRNA2-dCas9-HDAC1), it is recommended to combine both plasmids into the same precipitation tube to simplify handling for downstream procedures.
   4. The following day, centrifuge the DNA precipitation tubes at 11,000 × g for 15 min at 4 °C.
      NOTE: Perform all subsequent steps under sterile conditions in a biosafety cabinet, as this stage will also involve handling live cells.
   5. Carefully discard the supernatant. Add 500 µL of 70 % ethanol to wash the pellet. Centrifuge at 11,000 × g for 5 min at 4 °C.
   6. Remove the ethanol supernatant completely. Allow the pellet to air-dry until no visible ethanol remains.
   7. Resuspend the dried DNA pellet thoroughly in 50 µL of DMEM. Keep the plasmid-DMEM solution at 4 °C until use.
   8. Run 400 ng of the prepared episomal DNA (approximately 2.5 µL of the resuspended solution) on a 1% agarose gel to verify the success of DNA purification and integrity prior to transfection.
3. Prepare cells:
   1. Harvest K562 cells from a T-75 flask when cultures reach 50-60% confluency to ensure transfection efficiency.
   2. Count cells using a hemocytometer. Transfer a volume containing 2 × 10⁵ cells into a 35-mm culture dish containing 900 µL of DMEM without FBS or antibiotics.
      NOTE: Use serum-free DMEM for all steps in the transfection procedure. Resume full medium culture conditions only after the transfection incubation period is complete.
   3. Place the dish in the incubator (37 °C, 5% CO₂) to maintain the cells ready for transfection.
4. Use a Lipofectamine-based transfection kit for plasmid delivery. Follow the manufacturer's instructions for the specific kit used, adjusting volumes if needed.
   1. Add 3 µL of P3000 Reagent (supplied in the transfection kit) to the prepared plasmid-DMEM solution. Mix gently and incubate the plasmid-P3000 reagent mix at room temperature for 5 min.
   2. Add 5 µL of Lipofectamine 3000 Reagent (supplied in the transfection kit) along with enough DMEM to reach a total reaction volume of 100 µL. Incubate at room temperature for 15 min.
   3. Add the full 100 µL transfection mixture to the 35-mm dish containing cells. Gently shake the plate to disperse the mixture, then return the dish to the incubator.
5. At 24 h post-transfection, add 3 mL of full medium (DMEM supplemented with 10% FBS and 1× Pen/Strep) to each 35-mm dish to initiate proliferation of transfected cells. At 72 hours post-transfection, begin the selection procedure by adding G-418 antibiotic to a final concentration of 1 mg/mL.
6. Once selection is adequately established and the total cell count exceeds 1 × 10⁶, proceed to isolate single clones (Figure 3A) using fluorescence-activated cell sorting (FACS):
   1. Harvest the cells by centrifugation at 300 × g for 6 min at 25 °C. Wash once with 1× sterile phosphate-buffered saline (PBS, pH ~7.4) and centrifuge again under the same conditions.
   2. Resuspend the cell pellet in 1× PBS and determine the cell concentration using a hemocytometer. Dilute the suspension to 1 × 10⁶ cells/mL, ensuring thorough resuspension.
   3. Set up FCS shorter to single-cell mode (ensures 1 cell per droplet), using a 96-well tissue culture-treated plate as the collection device. Adjust software settings if needed, depending on the instrument used (Figure 3B).
   4. Gate the primary population based on expected size (FSC) and granularity (SSC). Exclude debris (low FSC/SSC) and highly granular or dead material (high SSC). Use FSC-A vs. FSC-H or SSC-A vs. SSC-H dot plots to gate singlets.
   5. Pre-fill the 96-well plate with 100 µL of full medium containing 1 mg/mL G-418 per well. Warm the plate in the incubator prior to sorting.
   6. Perform the sort in single-cell mode, maintaining a sorting rate below 300 events/second to enhance sorting accuracy.
   7. After sorting, inspect the wells under a microscope to confirm one cell per well. Place the plate in a 37 °C incubator with 5% CO₂.
7. Allow single-cell clones to expand individually in the 96-well plate. Once clonal growth is established, reduce G-418 concentration to 400 µg/mL to maintain selection.
8. Transfer well-growing clones sequentially to 12-well plates, then to T-25 and finally T-75 flasks as needed, until each clone reaches ~1 × 10⁶ cells.
9. Collect double cell pellets at regular intervals. Store a pellet at -20 °C for genomic DNA extraction. Additionally, store a pellet homogenized in 500 µL of Trizol Reagent at -20 °C for RNA extraction and cDNA synthesis.
   CAUTION: Trizol Reagent should be handled with caution due to its toxicity and corrosive nature in a chemical fume hood while wearing personal protective equipment, and contaminated materials must be labeled for hazardous waste disposal.
10. Extract genomic DNA from selected clones using traditional phenol-chloroform extraction, followed by ethanol precipitation.
    CAUTION: Phenol:chloroform is toxic and corrosive, and should be handled in a chemical fume hood while wearing personal protective equipment. Dispose phenol- and chloroform-containing waste as hazardous chemical waste.
11. Ensure DNA is fully resuspended in nuclease-free water or TE buffer. Measure DNA concentration and purity using a spectrophotometer before proceeding to downstream applications.
    NOTE: This DNA will be used in downstream experiments such as PCR and Pyrosequencing to assess epigenetic editing efficiency.
12. Extract total RNA from cell pellets using Trizol Reagent, followed by isopropanol precipitation²⁹. Handle RNA samples with caution by ensuring RNase-free reagents and disposables, and maintaining samples at 4 °C during the procedure.
    NOTE: Total RNA will be used for cDNA synthesis using a reverse transcription kit.

4. PCR to confirm the presence of both episomes in clones

1. Harvest approximately 5 × 10⁵ cells from each culture clone, after resuspending cells thoroughly. Centrifuge at 7,000 x g for 3 min at 25 °C. Discard supernatant medium.
2. Wash the cell by resuspending in 1× PBS. Centrifuge again under the same conditions.
3. Discard the supernatant, leaving a small volume, just above the pellet level. Use 1 µL of the cell suspension directly as a template for PCR.
4. Alternatively, use 200 ng of purified genomic DNA extracted from the transfected clones.
   NOTE: Direct PCR from washed cells provides a rapid screening method but may yield variable results. For higher specificity and sensitivity, use purified genomic DNA as the template.
5. Prepare the PCR master-mix according to Table 1.
6. Use two sets of primers, with a common forward primer targeting the dCas9 sequence and separate reverse primers specific to each epigenetic effector vector (Supplementary Table 3).
7. Follow the cycling conditions presented in Table 2.
8. Run PCR products on agarose gel or analyze with capillary electrophoresis to verify product sizes: 230 bp for the DNMT3A-containing vector and 205 bp for the HDAC1-containing vector (Figure 3C). Include a positive control (mix of both plasmids at a concentration of 10-20 ng) to confirm the expected band sizes and validate PCR efficiency. Include a no template control (NTC).

Reagent	Final concentration/amount
10× Buffer	1×
Forward primer	1 μΜ
Reverse primers	0.5 μM
DNA	200 ng
Polymerase	1.25 U
dNTPs	200 μM each
Water, nuclease-free	to 50 μL

Table 1: PCR reaction mix for episome verification.

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	30
Annealing	58	30 s
Extension	72	1 min
Final Extension	72	5 min	1

Table 2: PCR cycling conditions for episome verification.

5. Targeted pyrosequencing analysis

1. Treat gDNA (1.5 µg) extracted from transfected clones with a bisulfite conversion kit according to the manufacturer's instructions.
2. Design primers flanking the CpG sites within the regulatory region targeted by the constructs. Use a dedicated bisulfite primer design tool. Follow these design guidelines:
   1. Convert the CpG island or CpG site regulatory region to the bisulfite converted version.
   2. Design a forward and reverse primer for PCR amplification of bisulfite-converted DNA. Include a biotin label on one primer (usually the reverse) for immobilization on streptavidin-coated beads.
   3. Design a sequencing primer (18-25 bp) internal to the PCR product, upstream of the first CpG of interest. As a target region, select a 100 bp window upstream or downstream of the sgRNA binding site to assess potential local epigenetic modifications.
   4. Avoid CpG sites within primer sequences to ensure unbiased amplification.
   5. Choose a primer pair with an Assay Design software score greater than 75 % and a quality rating of 'high' (blue), which indicates that the software analysis did not identify any problems or concerns.
      NOTE: For primer sequences designed to target CpG island 326, consult Supplementary Table 3. The primers were designed to bind in regions flanking the sgRNA1 and sgRNA2 binding sites, allowing sequencing of CpG sites surrounding the targeted loci.
3. Set up a PCR reaction using a high-fidelity hot-start DNA polymerase optimized for bisulfite templates.
4. Pyrosequencing procedure:
   1. Bind biotinylated PCR products to streptavidin-coated sepharose beads and isolate single-stranded DNA using a pyrosequencing workstation.
   2. Add the sequencing primer to the single-stranded DNA in the annealing buffer. Heat the mixture to 80 °C for 2 min, then allow it to cool gradually to room temperature.
   3. Perform sequencing reactions using the pyrosequencing instrument and the dispensation order defined during primer design.
5. Data analysis:
   1. Use dedicated pyrosequencing software to quantify the percentage methylation at each CpG site.
   2. Compare methylation levels between transfected and untransfected clones. Assess editing efficiency (Figure 3D). Consider performing a two-tailed unpaired t-test to compare methylation levels between untreated controls and each transfected clone at each individual CpG site.

6. cDNA synthesis and digital PCR for analysis of dCas expression

1. Prepare total RNA (1,100 ng per reaction) extracted from transfected clones in 12 µL of RNase-free water. Keep samples on ice.
2. Assemble the genomic DNA elimination mix on ice: Mix 2 µL of gDNA wipeout buffer, RNA sample containing 1,100 ng of total RNA, and RNase-free water to a final volume of 14 µL. Incubate for 2 min at 42 °C, then place on ice.
3. Prepare the reverse transcription master mix on ice: Mix 4 µL of RT Buffer with 1 µL of RT primers, and add 1 µL of reverse transcriptase.
4. Combine 14 µL of genomic DNA elimination mix with 6 µL of reverse transcription master mix to a total reaction volume of 20 µL. Mix gently, and incubate at 42 °C for 15 min. Inactivate the reverse transcriptase by heating at 95 °C for 3 min.
   NOTE: Store cDNA at -20 °C until further use.
5. Dilute cDNA from all samples 1:1 with nuclease-free water. Use the diluted cDNA for downstream digital PCR analysis, using a digital PCR system with dCas-specific primers (Supplementary Table 3):
   1. Prepare the 4× Master mix and Reaction mix according to Table 3. Add diluted cDNA to the mix and pipette gently to ensure thorough mixing.
      NOTE: Set up reactions in at least duplicates for all clones. Include a negative control (cDNA from untreated K562 cells) and an NTC.
   2. Load 9 µL of the Reaction mix into each well of the PCR plate. Set up the PCR system using the cycling conditions presented in Table 4.
   3. Select the FAM dye channel for detection and start the run.
   4. After completion, retrieve the samples and analyze the data.
      NOTE: Digital PCR reports results as copies per µL of reaction.
   5. Multiply the reported value (copies/µL) by the reaction volume (10 µL) to obtain total copies per reaction. Divide this value by the amount of cDNA in the reaction to obtain copies per ng cDNA (equivalent to ng RNA input).
      NOTE: If the exact protocol is followed, each sample begins with 1,100 ng of RNA in a 20 µL RT reaction (55 ng/µL). After 1:1 dilution, the working concentration is 27.5 ng/µL, and since 1 µL is added to each PCR reaction, this corresponds to 27.5 ng of cDNA (RNA-equivalent) per reaction.

Master mix (4×)
Reagent	Final concentration/amount
25× SYBR Green dye	2×
5× Digital PCR mix	4×
Water, nuclease-free	to 100 μL
Reaction mix
Reagent	Final concentration/amount
4× Master mix	1×
Forward primer	1 μΜ
Reverse primer	1 μΜ
cDNA (diluted)	1 μL
Water, nuclease-free	to 10 μL

Table 3: 4× Master mix and reaction mix for digital PCR analysis of dCas expression.

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	96	10 min	1
Denaturation	96	5 s	40
Annealing/Extension	60	15 s

Table 4: Cycling conditions for digital PCR.

Results

To investigate whether our targeted epigenetic editing tools could induce site-specific DNA methylation changes at the ZBTB7A CpG island 326, we first generated episomal vectors encoding dCas9 fused to either DNMT3A(CD) or HDAC1, along with various sgRNAs designed with target sites within CpG island 326. We initially co-transfected K562 cells with combinations of these plasmids to evaluate whether simultaneous delivery of sgRNA and effector modules could lead to reproducible epigenetic modifications.

Discussion

One of the most critical steps in this protocol is the design of highly specific sgRNAs. Off-target effects are a known limitation of CRISPR-based epigenetic editing systems, which can often arise from dCas9 binding infidelity, as well as the non-specific activity of the effector domains. To minimize this, sgRNAs must be designed using bioinformatic tools that optimize for high on-target activity and minimal off-target binding³⁰. Employing design tools and validating predicted off-target sites is ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute Q DNA Digital PCR Master Mix (5x)	Applied Biosystems	A52490
AgeI-HF	NEB	R3552
AscI	NEB	R0558
AseI	NEB	R0526
BamHI-HF	NEB	R3136
BD FACS Melody Cell Sorter	BD
Bglll	NEB	R0144
BspEI	NEB	R0540
Deoxynucleotide (dNTP) Solution Mix (10 mM each)	NEB	N0447
DMEM High Glucose w/stable Glutamine, w/Sodium Pyruvate medium	Biowest	L0103
DNA Polymerase I, Large (Klenow) Fragment	NEB	M0210
DNMT3A (CD), HDAC1 sequences	IDT
DreamTaq DNA Polymerase	Thermo Scientific	EP0712
EpiTect	Qiagen	59104	Bisulfite conversion kit
Epitect Bisulfite kit	Qiagen	59104
Esp3I	NEB	R0734
Fetal Bovine Serum	Gibco	A5256701
Geneticin selective antibiotic (G418 sulfate)	Gibco	11811031
Lipofectamine 3000 transfection kit	Invitrogen	L3000015
NEB® 5-alpha Competent E. coli (High Efficiency)	NEB	C2987I
NEB® 5-alpha F'Iq Competent E. coli (High Efficiency)	NEB	C2992H
Nucleospin Tissue	Macherey-Nagel	740952.50
PBS, 10x solution pH 7.4	Lonza	BE17-517Q
Penicillin-Streptomycin solution 100x	Biowest	L0022
pGuide vector	OriGene Technologies	GE100042
Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA	Sigma-Aldrich	P2069
Primers, sgRNAs	Eurofins
PyroMark Assay Design	Qiagen	Version 2.0	Bisulfite primer design tool
PyroMark Gold Q24 Reagents	Qiagen	970802
PyroMark PCR kit	Qiagen	978703
PyroMark Q24 MDx	Qiagen		Pyrosequencing workstation
PyroMark Q24 Software	Qiagen	v2.0.8	Pyrosequencing software
Q5® High-Fidelity DNA Polymerase	NEB	M0491
QuantiTect Reverse Transcription Kit	Qiagen	205311
QuantStudio Absolute Q MAP16 Plate Kit	Applied Biosystems	A52865	Digital PCR system
SYBR Green I Nucleic Acid Gel Stain, 10,000x concentrate in DMSO	Invitrogen	S7563
T4 DNA Ligase	NEB	M0202
T4 Polynucleotide Kinase	NEB	M0201
TRIzol Reagent	Invitrogen	15596026