Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.
|Zinc Finger Nucleases: Highly-specific Genomic Scissors
|Figure 1: Each Zinc Finger Nuclease (ZFN) consists of two functional domains: a.) A DNA-binding domain comprised of a chain of two-finger modules, each recognizing a unique hexamer (6 bp) sequence of DNA. Two-finger modules are stitched together to form a Zinc Finger Protein, each with specificity of ≥ 24 bp. b.) A DNA-cleaving domain comprised of the nuclease domain of Fok I. When the DNA-binding and DNA-cleaving domains are fused together, a highly-specific pair of 'genomic scissors' are created.
Double-strand breaks are important for site-specific mutagenesis in that they stimulate the cell's natural DNA-repair processes, namely homologous recombination and Non-Homologous End Joining (NHEJ). By implementing established, field proven methods, these processes are harnessed to generate precisely targeted genomic edits, resulting in cell lines with targeted gene deletions, integrations, or modifications.
- Rapid disruption of, or integration into, any genomic loci
- Mutations made are permanent and heritable
- Works in a variety of mammalian somatic cell types
- Edits induced through a single transfection experiment
- Knockout or knock-in cell lines in as little as two months
- Single or biallelic edits occur in 1–20% of clone population
- No antibiotic selection required for screening
- Functional Genomics/Target Validation
- Creation of gene knockouts in multiple cell lines
- Complete knockout of genes not amenable to RNAi
- Cell-based screening
- Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes
- Cell Line Optimization
- Creation of cell lines that produce higher yields of proteins or antibodies
|ZFN-Mediated Targeted Genome Editing
|Figure 2: ZFN-mediated genome editing takes place in the nucleus when a ZFN pair targeting the user’s gene of interest is delivered into a parental cell line, either by transfection, electroporation or viral delivery.
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