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

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.