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 (although sequences with a single mismatch occur about three times per human-sized genome). 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.

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 encoded 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 encodes meganucleases 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. The most widespread and best known is the LAGLIDADG family. LAGLIDADG family endonucleases are mostly found in the mitochondria and chloroplasts of eukaryotic unicellular organisms.

The name of this family 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.