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Why ZFNs Are Less Common in Genome Editing

Zinc finger nucleases, or ZFNs, are one of the three functional technologies used in genome editing. Along with transcriptional activators like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), ZFNs are used to manipulate genomic sequences for the purpose of studying human disease and making advances in the field of human gene therapy.

What are ZFNs and How Do They Work?

Basically, ZFNs are artificially generated structures resulting from the fusion of a custom-designed DNA-binding zinc finger protein and Fok1 restriction endonuclease (the DNA-cleavage domain).

ZFNs have three to six zinc finger domains, or repeats (each containing about 30 amino acid residues) capable of recognizing between three to six nucleotide triplets or 9 and 18 base pairs each. While ZFNs do not usually exist in dimeric form, they dimerize upon recognizing the binding site.

On the other hand, the DNA-cleavage domain, which usually consists of Fok1 restriction enzyme, is responsible for cutting the DNA at a specific codon (usually within five to seven base pairs between two flanking zinc-finger binding sites). Upon the fusion of the DNA-binding and DNA-cleavage domains, a highly specific pair of genomic scissors is produced.

Here’s how it works.

1. ZFNs are introduced transiently into the cell by transfection or electroporation.
2. The DNA-binding and DNA-cleavage domains are released to enter the nucleus.
3. Upon recognizing the target site, the protein-binding domain heterodimerizes and binds adjacent sites on the target DNA with precise sequence specificity and spacing, thereby ensuring that cleavage occurs at the desired cleavage site.
4. The DNA-cleavage domain (Fok1) introduces a double-stranded DNA break to cut out the identified DNA segment. These double-strand breaks play an important role, since they stimulate the cell’s natural DNA-repair processes (i.e., homologous recombination and non-homologous end-joining or NHEJ) which help generate the desired genomic edits (i.e., gene deletion, integration, and modification).
5. The desired DNA segment is inserted into the DNA sequence.

Note: In the absence of a repair template, DNA repair proceeds through NHEJ. However, if the ZFN pair is co-transfected with a repair template, the frequency of homologous recombination would be more than a thousand times higher, and more than 20% of the cells will contain the gene insertion at the target site.

Benefits and Applications

Discovered in 1990, the technology is recognized as the first targeted nuclease to gain widespread use and has gained immense popularity due to its simplicity and specificity. Why do researchers use the ZFN method? Here are some reasons why.

• It facilitates the rapid disruption or integration into any genomic loci.
• It is applicable in a variety of mammalian somatic cell types.
• It can produce knockout or knock-in cell lines in a short period.
• Edits can be easily induced.
• It can produce permanent and heritable mutations.
• Antibiotic selection is not required for screening.

ZFNs can be used to manipulate the genomes of higher organisms (plants and animals). Specifically, they can be used for the following applications:

• Functional genomics or target validation. ZFNs can be used to create gene knockouts in multiple cell lines.
• Cell-based screening. ZFNs allow for the insertion of specific genes, promoters, fusion tags, or reporters into endogenous genes.
• Cell line optimization. ZFNS can also be used for priming cell lines for optimum performance (higher yield, etc.)

Limitations

Despite the apparent advantages, the technology has several limitations.

• It has an extremely high ratio of off-target mutations and cleavages. The technology is prone to errors, since each zinc finger repeat can only recognize three base pairs. As a result, mutations may occur at non-specific sites that exhibit similarity with the target sequence.
• It may cause immunogenicity. Since ZFNs are foreign bodies, they may provoke a humoral or cell-mediated immune response in the host organism.
• It lacks the desired flexibility. Unlike other more recent genome-editing technologies, such as TALENs and CRISPR-Cas9, it can be difficult to produce ZFNs that effectively recognize all DNA triplets.

For these reasons, ZFNs are less common in modern genome editing than more recent methods.