Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

The zinc finger nuclease or ZFN contains a zinc-ion stabilized, DNA-binding macro-domain connected to an endonuclease domain via a linker. This structure helps maintain specificity while cutting DNA.

To use ZFNs for genome editing, take a culture of human pluripotent stem cells. Supplement it with a plasmid encoding ZFN. Add a repair plasmid containing the target gene and the antibiotic resistance gene sandwiched between homology arms or HAs. Electroporate the cell-DNA mixture where the electric current facilitates the entry of plasmids inside the cell.

Once inside, the zinc finger motifs of translated ZFN bind to the triplets of complementary base pairs in the host genome, correctly positioning the Fok-1 restriction endonuclease at the target site. ZFNs bind to opposite strands in pairs allowing Fok-1 homodimerization to form an active catalytic center in which Fok-1 generates DNA double-stranded breaks or DSBs, producing a four-nucleotide overhang.

The presence of HAs in repair plasmid guides the homology-dependent repair where the overhanging end inverts and uses the homologous HA sequence as a template. The DNA synthesis continues by adding nucleotides complementary to the target gene.

The resulting gaps are ligated, facilitating gene insertion. Additionally, the promoterless antibiotic resistance gene is expressed from a host promoter upstream of the insertion site. Successfully edited cells grow in the antibiotic selection medium.

Begin by culturing human pluripotent stem cells in hESC media on a 6-well plate containing mitomycin C-inactivated Mouse Embryonic Fibroblast, or MEF, feeder cells grown on gelatin. Each subsequent day after plating, until the hPSC has reached 50% confluency, use a glass pipette and vacuum to remove the entire volume of media. Replace with 3 milliliters of warm hESC media per well.

One day before targeting, remove the hESC media, and add fresh pre-warmed hESC media supplemented with 10 micromolar Y27632. Also, prepare one to two 6-well plates of drug-resistant MEF feeder cells from DR4 mice. On the day of targeting, prepare the transfection solutions by pipetting 5 micrograms of zinc finger nuclease expression plasmid 1 and 2 into a 1.5-milliliter tube.

Add 30 micrograms of the repair donor plasmid, followed by enough 1X Phosphate Buffered Saline to bring the volume to 300 microliters. Inspect the cells under a microscope to ensure 50% confluency. Next, use a glass pipette and vacuum to remove the media from the plate of hPSCs. Then, wash the cells with 2 milliliters of warm 1X PBS. After aspirating the PBS, add 0.5 milliliters of 0.25% trypsin EDTA solution, directly onto the cells.

Place in the tissue culture incubator for approximately 10 minutes, or until the feeder layer begins to lift off the plate. Following the incubation, add 2 milliliters of warm esWash media to each well to stop the trypsin reaction. Collect the cells from each well, ensuring that the feeder cells come off as a sheet. Pipette the contents of each well into a single 50-milliliter conical tube, combining all wells, and triturate the cells using a 10-milliliter serological pipette.

Add esWash media to bring the cell suspension to 40 milliliters. Wait one to two minutes to allow large feeder chunks to settle at the bottom of the tube, then, remove the supernatant using a serological pipette, and deposit into a fresh 50-milliliter conical tube. After centrifuging the cell suspension for five minutes at 190 x g, aspirate the supernatant without disturbing the cell pellet. Resuspend the cells in 500 microliters of 1X PBS.

Combine the resuspended cells with the plasma transfection solution prepared earlier. Pipette the cells and transfection mixture into a 4-millimeter electroporation cuvette, and place on ice for three to five minutes. Set the parameters for the exponential program on the electroporation system to 250 volts, 500 microFarads, infinite resistance, and 4-millimeter cuvette size. Electroporate the cells, and then, place the cuvette back on ice for three minutes.

Resuspend the electroporated cells in 18 milliliters of warm hESC media supplemented with 10 micromolar Y27632. Inspect the DR4 MEFs under a microscope. Plate 3 milliliters of the single-cell suspension into each well of a 6-well plate containing DR4 feeder cells, and return to the incubator. On day three after plating, replace the media on the cells with hESC media without Y27632. On day 4, replace the unsupplemented media with media containing the appropriate selection antibiotic. Puromycin is used here.