There are three families of engineered nucleases being used:
|1||Zinc finger nucleases (ZFNs)|
|2||Transcription Activator-Like Effector-based Nucleases (TALENs)|
DNA double-stranded break (DSB) repair mechanisms
To understand these concepts you need to understand the concept of DNA double-stranded break (DSB) repair mechanisms. Two of the known DSB repair pathways that are essentially functional in all organisms are the non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends in a double-strand break. In contrast, in HDR, a homologous sequence is utilized as a template for the regeneration of missing DNA sequences at the breakpoint. The natural properties of these pathways form the very basis of nucleases based genome editing.
NHEJ is error-prone and has been shown to cause mutations at the repair site. Thus if one is able to create a DSB at the desired gene in multiple samples, it is very likely that mutations will be generated at that site in some of the treatments because of errors created by the NHEJ infidelity. On the other hand, the dependency of HDR on a homologous sequence to repair DSBs can be exploited by inserting a desired sequence within a sequence that is homologous to the flanking sequences of a DSB which, when used as a template by HDR system, would lead to the creation of the desired change within the genomic region of interest.
Despite the distinct mechanisms, the concept of the HDR based gene editing is in a way similar to that of homologous recombination-based gene targeting. However, the rate of recombination is increased by at least three orders of magnitude when DSBs are created and HDR is at work thus making the HDR based recombination much more efficient and eliminating the need for stringent positive and negative selection steps. So based on these principles if one is able to create a DSB at a specific location within the genome, then the cell’s own repair systems will help in creating the desired mutations.
Site-specific double-stranded breaks
The creation of a DSB in DNA is easy by using restriction enzymes. However, if genomic DNA is treated with a particular restriction endonuclease many DSBs will be created. This is a result of the fact that most restriction enzymes recognize a few base pairs on the DNA as their target and very likely that particular base-pair combination will be found in many locations across the genome. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALEN) and meganucleases.