ZFN & TALEN on ZAGENO
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In nature, nucleases (a type of restriction enzyme) are utilized by cells for DNA repair and proofreading by cleaving bonds between nucleic acids. By artificially engineering nucleases in the lab, scientists can harness the power of restriction enzymes to slice DNA strands at desired locations, initiating DNA repair pathways that integrate modified DNA strands into the genome. Two of the most common techniques of artificially engineered nucleases are Zinc-fingered nuclease (ZFN) and Transcription Activator-Like Effector Nucleases (TALEN).
Zinc Finger Nucleases (ZFN)
Zinc Finger Nucleases are a combination of a zinc finger DNA-binding domain and a DNA cleavage domain. The DNA-binding domain is composed of three to six zinc finger repeats which recognize between 9 to 18 base pairs. The DNA cleavage domain contains the endonuclease Fokl due to its non-specific cleavage. Do note that Fokl must form a dimer to cleave DNA, so a pair of ZFN’s is necessary for gene editing.
ZFN is particularly useful in manipulating genomes from a wide range of plants and animals, disable alleles using non-homologous end-joining, can serve as a potential treatment for HIV/AIDS, edit alleles using homologous recombination, and is employed in the creation of isogenic human disease models.
Problems with ZFN occur with off-target cleavage and immunogenicity, which happens when ZFN’s activate an immune response.
Transcription Activator-Like Effector Nucleases are similar to ZFN’s in that they are two domains fused together. However, instead of a Zinc Finger DNA-Binding Domain, TALEN uses a TAL effector DNA-binding domain joined with a Fokl-based DNA cleavage domain. TAL effectors are 33 – 34 amino acid long proteins, with differing 12th and 13th amino acids known as Repeat Variable Diresidues. These two positions alone are what allow for TALEN to have specific nucleotide recognition.
Once the TALEN complex has been constructed, they are delivered into the cell either through plasmids or mRNA. There, TALENs are particularly useful for modifying plant genomes, biofuel development, stem cell engineering, gene therapies, and introducing knockouts into the genome.