Presently, there are several companies and universities, which are exploring the potential of different gene editing technologies beyond CRISPR for basic research, and the development of gene editing solutions. With the development of a variety of versatile DNA modulation technologies such as include zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), engineered endonucleases / meganucleases (EMNs) and clustered regularly interspaced short palindromic repeats (CRISPR), genetic engineering and genome editing 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 technologies has opened up the possibility of modifying genomic sequences in both eukaryotic and prokaryotic organisms. 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.
The history of some gene editing techniques is highlighted in the figure below.
Type of Genome Editing:
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; 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.
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 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.
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 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
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