Since the discovery of DNA in the 1950s, researchers aim to understand this universal code of life. On the one hand, basic science wants to decipher fundamental phenomena and mechanisms. On the other hand, the applied sciences, such as biotechnology or medicine, try to use this information to create more robust crops or cure disease.
A common method of all researchers interested in DNA and the associated phenotype is modification. To find out the role of a certain gene and its respective proteins (via gene expression), an obvious strategy is to get rid of it (knock-out) or modify it (mutation) and watch the result. This goal is why over the years several techniques were developed to alter genome information.
The use of bacterial derived restriction enzymes (EcoRI, BamHI, HindIII etc.) to cut DNA at specific sites is one example of a gene altering method. These enzymes are used for molecular cloning, i.e., to fuse recombinant DNA for expression in a host organism. Each restriction enzyme has only one specific DNA sequence it can cut. Ligases fuse the targeted DNA piece into a plasmid vector. The cloning process takes place in vitro and the recombinant DNA plasmid has to be transferred back into the cell to produce the relevant protein.
A way to manipulate genes in their endogenous state, meaning directly in the target cell’s inherent genome, was developed due to the discovery of homologous recombination (HR). Exogenous DNA fragments can be introduced to the target genome by making use of sequence homology. In a few cases, a DNA fragment showing homology to the target sequence can integrate into the genome by recombination (1 in 106-109 cells). This discovery facilitated the creation of such things as knock-in (one or more genes added) and knock-out animals.
Despite these advances, the rare event of homologous recombination called for improvement. Luckily further ways to manipulate DNA came to light. It was discovered that another type of nucleic acid cleaving enzymes – nucleases – could be programmed and used for genetic manipulation. At first three major protein classes were exploited. Meganucleases derived from bacteria, zinc finger nucleases (ZFN) based on eukaryotic transcription factors, and transcription activator-like effector nucleases (TALENs) from bacteria all recognize their DNA binding site by protein-DNA interaction. Meganucleases have a DNA binding domain and a nuclease domain per se, whereas ZFs and TALEs are attached to a FokI nuclease domain.
In principle, genome editing techniques based on meganucleases, ZFN, or TALENs also use homologous recombination to introduce a piece of exogenous DNA, but they don't wait for a double strand break to occur by accident. Due to their sequence specificity and nuclease activity, they are able to produce targeted DNA double strand breaks (