Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-SceI meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
Among meganucleases, the LAGLIDADG family of homing endonucleases has become a valuable tool for the study of genomes and genome engineering over the past fifteen years. Meganucleases are "molecular DNA scissors" that can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases are used to modify all genome types, whether bacterial, plant or animal. They open up wide avenues for innovation, particularly in the field of human health, for example the elimination of viral genetic material or the "repair" of damaged genes using gene therapy.
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Meganucleases are found in a large number of organisms – Archaea or archaebacteria, bacteria, phages, fungi, yeast, algae and some plants. They can be expressed in different compartments of the cell – the nucleus, mitochondria or chloroplasts. Several hundred of these enzymes have been identified.
Meganucleases are mainly represented by two main enzyme families collectively known as homing endonucleases: intron endonucleases and intein endonucleases.
In nature, these proteins are coded by mobile genetic elements, introns or inteins. Introns propagate by intervening at a precise location in the DNA, where the expression of the meganuclease produces a break in the complementary intron- or intein-free allele. For inteins and group I introns, this break leads to the duplication of the intron or intein at the cutting site by means of the homologous recombination repair for double-stranded DNA breaks.
We know relatively little about the actual purpose of meganucleases. It is widely thought that the genetic material that codes for them functions as a parasitic element that uses the double-stranded DNA cell repair mechanisms to its own advantage as a means of multiplying and spreading, without damaging the genetic material of its host.
There are five families, or classes, of homing endonucleases[1]. The most widespread and best known is the LAGLIDADG family. It is mostly found in the mitochondria and chloroplasts of eukaryotic unicellular organisms.
Its name corresponds to an amino acid sequence (or motif) that is found, more or less conserved, in all the proteins of this family. These small proteins are also known for their compact and closely packed three-dimensional structures.
The best characterized endonucleases which are most widely used in research and genome engineering include I-SceI (discovered in the mitochondria of baker's yeast Saccharomyces cerevisiae), I-CreI (from the chloroplasts of the green algae Chlamydomonas reinhardtii) and I-DmoI (from the archaebacterium Desulfurococcus mobilis).
The best known LAGLIDADG endonucleases are homodimers (for example I-CreI, composed of two copies of the same protein domain) or internally symmetrical monomers (I-SceI). The DNA binding site, which contains the catalytic domain, is composed of two parts on either side of the cutting point. The half-binding sites can be extremely similar and bind to a palindromic or semi-palindromic DNA sequence (I-CreI), or they can be non-palintromic (I-SceI).
The high specificity of meganucleases gives them a high degree of precision and much lower cell toxicity than other naturally occurring restriction enzymes; they were identified in the 1990s as particularly promising tools for genome engineering.
However, the meganuclease-induced genetic recombinations that could be performed were limited by the repertoire of meganucleases available. Despite the existence of hundreds of meganucleases in nature, and the fact that each one is able to tolerate minor variations in its recognition site, the probability of finding a meganuclease able to cut a given gene at the desired location is extremely slim. Several research laboratories therefore soon began trying to engineer new meganucleases targeting the desired recognition sites.
The most advanced research and applications concern homing endonucleases from the LAGLIDADG family.
To create tailor-made meganucleases, two main approaches have been adopted:
These two approaches can be combined to increase the possibility of creating new enzymes, while maintaining a high degree of efficacy and specificity. The scientists from Cellectis, a French biotechnology company, have developed a collection of over 20,000 protein domains from the homodimeric meganuclease I-CreI as well as from other meganucleases scaffolds[8]. They can be combined to form functional chimeric tailor-made heterodimers for research laboratories and for industrial purposes.
Precision Biosciences, an American biotechnology company, has developed a fully rational design process called Directed Nuclease Editor (DNE) which is capable of creating engineered meganucleases that target and modify a user-defined location in a genome[9].
Explanatory video about meganucleases, by Cellectis