Name: Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins (Video) | JoVE Journal
Uploaded: 2025-10-31T14:30:00.000+00:00
Duration: 9 min 56 s
Description: Learn a dual-vector episomal strategy for stable targeted DNA methylation in K562 cells using dCas9-DNMT3A and dCas9-HDAC1 fusion proteins. This method enables precise epigenetic editing without genome integration.

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5 minutes

Irene Dereki and Vasiliki Chondrou are supported by a three-year fellowship (Grant No. 80706) from the Special Account of Research Funds (ELKE) of the Hellenic Open University.

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The authors declare no competing financial or non-financial interests.

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 ...

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 syndromes1,2,3. 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 developed6,7,8,9. 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 phenotypes10,11,12.
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 tools13,14. 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&#8239;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 &#34;hit-and-run&#34; 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 effectors17,18,19.
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 overexpression20,21. The Scaffold/Matrix Attachment Region (S/MAR) element of the episome interacts with the nuclear matrix, contributing to the episome&#39;s stable maintenance in the cells&#39; nucleus during cell division22,23. 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&#39; 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 approaches19,24. 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.

<table><tbody><tr><td>Absolute Q DNA Digital PCR Master Mix (5x)</td><td>Applied Biosystems</td><td>A52490</td><td></td></tr><tr><td>AgeI-HF&amp;nbsp;</td><td>NEB</td><td>R3552</td><td></td></tr><tr><td>AscI</td><td>NEB</td><td>R0558</td><td></td></tr><tr><td>AseI</td><td>NEB</td><td>R0526</td><td></td></tr><tr><td>BamHI-HF</td><td>NEB</td><td>R3136</td><td></td></tr><tr><td>BD FACS Melody Cell Sorter</td><td>BD</td><td></td><td></td></tr><tr><td>Bglll</td><td>NEB</td><td>R0144</td><td></td></tr><tr><td>BspEI</td><td>NEB</td><td>R0540</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix (10 mM each)</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>DMEM High Glucose w/stable Glutamine, w/Sodium Pyruvate medium</td><td>Biowest</td><td>L0103</td><td></td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>NEB</td><td>M0210</td><td></td></tr><tr><td>DNMT3A (CD), HDAC1 sequences</td><td>IDT</td><td></td><td></td></tr><tr><td>DreamTaq DNA Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>EP0712</td><td></td></tr><tr><td>EpiTect</td><td>Qiagen</td><td>59104</td><td>Bisulfite conversion kit&amp;nbsp;</td></tr><tr><td>Epitect Bisulfite kit</td><td>Qiagen</td><td>59104</td><td></td></tr><tr><td>Esp3I</td><td>NEB</td><td>R0734</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>Geneticin selective antibiotic (G418 sulfate)</td><td>Gibco</td><td>11811031</td><td></td></tr><tr><td>Lipofectamine 3000 transfection kit</td><td>Invitrogen</td><td>L3000015</td><td></td></tr><tr><td>NEB&amp;reg;&amp;nbsp;5-alpha Competent&amp;nbsp;E. coli&amp;nbsp;(High Efficiency)</td><td>NEB</td><td>C2987I</td><td></td></tr><tr><td>NEB&amp;reg;&amp;nbsp;5-alpha F&#039;Iq&amp;nbsp;Competent&amp;nbsp;E. coli&amp;nbsp;(High Efficiency)</td><td>NEB</td><td>C2992H</td><td></td></tr><tr><td>Nucleospin Tissue&amp;nbsp;</td><td>Macherey-Nagel</td><td>740952.50</td><td></td></tr><tr><td>PBS, 10x solution pH 7.4</td><td>Lonza</td><td>BE17-517Q</td><td></td></tr><tr><td>Penicillin-Streptomycin solution 100x</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>pGuide vector</td><td>OriGene Technologies</td><td>GE100042</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA</td><td>Sigma-Aldrich</td><td>P2069</td><td></td></tr><tr><td>Primers, sgRNAs&amp;nbsp;</td><td>Eurofins</td><td></td><td></td></tr><tr><td>PyroMark Assay Design&amp;nbsp;</td><td>Qiagen</td><td>Version 2.0</td><td>Bisulfite primer design tool&amp;nbsp;</td></tr><tr><td>PyroMark Gold Q24 Reagents</td><td>Qiagen</td><td>970802</td><td></td></tr><tr><td>PyroMark PCR kit&amp;nbsp;</td><td>Qiagen</td><td>978703</td><td></td></tr><tr><td>PyroMark Q24 MDx</td><td>Qiagen</td><td></td><td>Pyrosequencing workstation&amp;nbsp;</td></tr><tr><td>PyroMark Q24 Software&amp;nbsp;</td><td>Qiagen</td><td>v2.0.8</td><td>Pyrosequencing software&amp;nbsp;</td></tr><tr><td>Q5&amp;reg; High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit</td><td>Qiagen</td><td>205311</td><td></td></tr><tr><td>QuantStudio Absolute Q MAP16 Plate Kit</td><td>Applied Biosystems</td><td>A52865</td><td>Digital PCR system&amp;nbsp;</td></tr><tr><td>SYBR Green I Nucleic Acid Gel Stain, 10,000x concentrate in DMSO</td><td>Invitrogen</td><td>S7563</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr></tbody></table>

epigenetic engineering of k562 cells: dual-vector episomal strategy for stable targeted dna methylation using dcas9-dnmt3a and -hdac1 fusion proteins

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

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&#160;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&#39;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.

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.
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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.

Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns ...

Our research aims to develop a stable, non-integrated system for targeted DNA methylation, exploring durable epigenetic editing in K562 cells. Recent advance in the field include dual-effector CRISPR-dCas9 systems, combining epigenetic enzymes for synergistic, stable, and local-specific epigenetic modification. To begin, maintain K562 cells in T75 culture flasks at 37 degrees Celsius with 5%carbon dioxide and humidified conditions.Quantify the episomal DNA to be used for transfection using a spectrophotometer at 260 nanometers. Use eight micrograms of total plasmid DNA per transfection by preparing four micrograms of each plasmid for a co-transfection, and mix the required amount with 90 microliters of ultrapure water. Now, add 10 microliters of 3 Molar sodium acetate solution and 2.5 volumes of 100%ethanol to the diluted DNA solution.Mix the DNA solution thoroughly and incubate it overnight at 20 degrees Celsius to allow DNA precipitation. On the following day, centrifuge the DNA precipitation tube at 11, 000 G for 15 minutes at four degrees Celsius. Under sterile conditions in a biosafety cabinet, carefully discard the supernatant from the tube.Add 500 microliters of 70%ethanol to the tube to wash the pellet, and centrifuge again at 11, 000 G for five minutes at four degrees Celsius. Next, remove the ethanol supernatant completely from the tube, and let the DNA pellet air dry until no visible ethanol remains. Then, re-suspend the dried DNA pellet thoroughly in 50 microliters of DMEM.Keep 400 nanograms of the re-suspended DNA approximately 2.5 microliters in a new tube, and store the plasmid DMEM solution at four degrees Celsius until further use. Run the remaining DNA sample on a 1%agarose gel to verify DNA purification and integrity. Harvest K562 cells from a T75 flask when cultures reach 50 to 60%confluency to ensure optimal transfection efficiency.Count the harvested cells using a hemocytometer, and transfer a volume containing two times 10 to the power of five cells into a 35 millimeter culture dish containing 900 microliters of DMEM without FBS or antibiotics. Place the culture dish in a 37 degrees Celsius incubator with 5%carbon dioxide to maintain the cells in a ready state for transfection. Use a Lipofectamine-based transfection kit to deliver plasmids following the manufacturer's instructions and adjusting volumes if needed.Add three microliters of P3000 reagent to the previously prepared plasmid DMEM solution. Mix gently, and incubate the mixture at room temperature for five minutes. Now, add five microliters of Lipofectamine 3000 reagent along with enough DMEM to reach a final volume of 100 microliters.Incubate this transfection mix at room temperature for 15 minutes. Add the full 100 microliters of the transfection mixture to the 35 millimeter culture dish containing the cells. Gently shake the dish to disperse the mixture evenly, then return it to the incubator.At 24 hours post-transfection, add three milliliters of full medium DMEM supplemented with 10%FBS and one times penicillin streptomycin to each dish to promote proliferation of transfected cells. At 72 hours post-transfection, begin selection by adding G418 antibiotic to reach a final concentration of one milligram per milliliter. Once the selection process is established and total cell count exceeds one times 10 to the power of six, harvest the cells by centrifugation at 300 G for six minutes at 25 degrees Celsius.Wash the pellet once with one-time sterile PBS, pH approximately 7.4, and centrifuge again under the same conditions. After re-suspending the final cell pellet in one-times PBS, determine the cell concentration using a hemocytometer. Dilute the suspension to one times 10 to the power of six cells per milliliter, ensuring the cells are fully re-suspended, and transfer cells to a flow cytometry tube.Next, set up the fluorescence activated cell sorter to single cell mode using a 96-well tissue culture-treated plate as the collection device. Adjust the software settings depending on the instrument used. Gate the primary population by selecting cells based on forward scatter and side scatter to isolate expected size and granularity.Exclude debris and highly granular or dead cells, and use forward scatter area versus height or side scatter area versus height plots to gate singlets. Then, pre-fill each well of the 96-well plate with 100 microliters of full medium containing one milligram per milliliter of G418, and warm the plate in a 37 degrees Celsius incubator. Perform cell-sorting in single cell mode, keeping the sorting rate below 300 events per second to improve accuracy.After sorting, inspect each well under a microscope to confirm the presence of a single cell per well, and place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide. Allow the single cell clones to expand individually in the 96-well plate. Once clonal growth is evident, add medium containing reduced G418 to the wells to a final concentration of 400 micrograms per milliliter to maintain selection pressure.Transfer well-growing clones sequentially to 12-well plates, then to T25 flasks, and finally, to T75 flasks as needed until each clone reaches approximately one times 10 to the power of six cells. Next, collect double cell pellets at regular intervals. Store one pellet at minus 20 degrees Celsius for genomic DNA extraction.Homogenize a second pellet in 500 microliters of TRIzol reagent, and store it minus 20 degrees Celsius for RNA extraction and complimentary DNA synthesis. Extract genomic DNA from selected clones using phenol-chloroform extraction followed by ethanol precipitation. Fully re-suspend the extracted DNA in nuclease-free water or TE buffer.Measure the DNA concentration and purity using a spectrophotometer before continuing to downstream steps. Finally, extract total RNA from the stored cell pellets using TRIzol reagent, followed by isopropanol precipitation. Single cells were sorted into 96-well plates using fluorescence-activated cell-sorting to isolate those carrying both constructs.PCR analysis of 10 expanded clones confirmed that only some retained both constructs, indicated by bands at 230 base pairs and 205 base pairs. Pyrosequencing revealed that the positive clone two showed increased methylation at multiple CPG sites near the gRNA1 target region compared to the untransfected control. We address the lack of non-integrated long-term delivery systems, enabling precise, stable epigenetic editing without altering the host genome.Our findings establish a reliable tool for studying gene regulation via target methylation, and support developing possible precise epigenetic therapies. Future research will explore improving episomal delivery, minimizing of target effects, enhancing with Cas9 efficiency, and adapting the system for primary or in vivo models.

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Learn a dual-vector episomal strategy for stable targeted DNA methylation in K562 cells using dCas9-DNMT3A and dCas9-HDAC1 fusion proteins. This method enables precise epigenetic editing without genome integration.

Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins (Video) | JoVE Journal

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

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