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