Gene Editing Technology: ZFN, TALEN and Meganucleases

Presently, there are several companies and universities, which are exploring the potential of gene editing technology beyond CRISPR for basic research, and the development of gene editing solutions. With the development of a versatile gene editing technology, including zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), engineered endonucleases / meganucleases (EMNs) and clustered regularly interspaced short palindromic repeats (CRISPR), genetic engineering and gene editing technology concepts have evolved significantly over the last two decades.

Gene engineering based on recombination was pioneered in the mid-1990s; Currently, development of gene editing technology has opened up the possibility of modifying genomic sequences in both eukaryotic and prokaryotic organisms and emergence of various cell and gene therapy companies. Genome Editing is a way of making changes in the DNA. There are various genome editing technologies which use enzymes that recognize and attach on to specific sites. These technologies act as scissors, cutting the DNA at specific spots. They also allow genetic material to be added, removed, or altered at particular locations in the genome. This has developed a myriad of products associated with the gene therapy market

The history of gene editing technology is highlighted in the figure below.

the history of some gene editing techniques is highlighted in the figure below.
Bringing in Light Genome Editing Technology: ZFN, TALEN and Meganucleases

Type of Genome Editing Technology:

  1. Zinc-Finger Nuclease

The structure of ZFN includes:

  • The target site of the ZFN is recognized by the ” left ” and ” right ” monomers consisting of a tandem array of three to six engineered ZFPs
  • A single engineered ZFP can recognize a nucleotide triplet.
  • Each ZNF is linked to a nuclease domain from the FokI restriction enzyme.
ZFNs are first engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations
Bringing in Light Genome Editing Techniques: ZFN (Zinc-Finger Nuclease)

 ZFNs are first engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations; these have proved to be most versatile and effective. Zinc-finger nucleases (ZFNs) are one of the most efficient and effective tools for genome editing. These are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes.

Bringing in Light Genome Editing Techniques: ZFN, TALEN and Meganucleases
Genome Editing Technology – Science behind ZFN technique
  • TALENs

The structure of TALEN includes:

  • TALENs recognize single nucleotides rather than relying on 3-base pair sites .
  • TALE repeats, which can naturally occur, consist of 10-30 repeat tandem arrays that bind and identify longer DNA sequences
  • The repeats and base pairs in the target DNA sequences have a one-to-one connection.
TALENs recognize single nucleotides rather than relying on 3-base pair sites
Genome Editing Technology – structure of TALEN

TALENs are restriction enzymes which enable the targeted alteration of any DNA sequence in a wide range of cell types and organismsTALENs is a fusion of transcription activator-like (TAL) proteins with FokI nucleases (cleavage domain). The TAL protein consists of 33-34 amino acid repeat patterns, with two variable positions. They have strong ability to identify specific nucleotides. When two TALENs bind and meet, the FokI domains induce a double-strand break which can inactivate a gene or can be used to insert DNA of interest.

TALENs are restriction enzymes which enable the targeted alteration of any DNA sequence in a wide range of cell
Genome Editing Technology – science behind TALENs technique
  • Meganucleases

Meganucleases are characterized by their capacity to recognize and cut large DNA sequences (12-40 basepairs). LAGLIDADG homing endonucleases (“meganucleases”) are highly specific DNA cleaving enzymes that are used for genome engineering. The structure of meganucleases is highlighted in the figure below.

Meganucleases are characterized by their capacity to recognize and cut large DNA sequences (12-40 basepairs).
Genome Editing Technology – structure of Meganucleases

Meganucleases are sequence specific endonucleases, which allow deletion, insertion, correction and single-site mutation in a controlled manner. Meganucleases or homing endonucleases are efficient in cleaving dsDNA at specific sites of around 14–40 bp and are considered as most specific naturally occurring restriction enzymes. Meganucleases can replace, eliminate or modify any sequence of interest in a highly efficient and targeted manner since they are capable to alter their recognition sequence via protein engineering

Meganucleases are sequence specific endonucleases, which allow deletion, insertion, correction and single-site mutation in a controlled manner.
Genome Editing Technology – science behind Meganucleases technique

Owing to the various novel solutions offered by novel gene editing technology, we anticipate that the gene editing therapy market is going to witness a sound growth in the foreseen future.

For more details, you can also download the SAMPLE REPORT on gene editing technology by Roots Analysis.

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