Meganucleases
Encyclopedia
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.
or archaebacteria, bacteria, phages
, fungi, yeast
, algae
and some plants. They can be expressed in different compartments of the cell – the nucleus
, mitochondria
or chloroplast
s. Several hundred of these enzyme
s 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, intron
s or intein
s. 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.
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).
.
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. 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.
Endodeoxyribonucleases
Endodeoxyribonucleases are enzymes which are both deoxyribonucleases and endonucleases.They are classified with EC numbers 3.1.21 through 3.1.25.Examples include:* DNA restriction enzymes* micrococcal nuclease...
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
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. 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
Human genome
The human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA. 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining...
to be found once by chance. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
.
Among meganucleases, the LAGLIDADG family of homing endonucleases has become a valuable tool for the study of genomes and genome engineering
Genome engineering
Genome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms....
over the past fifteen years.
Meganucleases are "molecular DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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.
Two main families
Meganucleases are found in a large number of organisms – ArchaeaArchaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
or archaebacteria, bacteria, phages
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
, fungi, yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, algae
Algae
Algae are a large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms, such as the giant kelps that grow to 65 meters in length. They are photosynthetic like plants, and "simple" because their tissues are not organized into the many...
and some plants. They can be expressed in different compartments of the cell – the nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, mitochondria
Mitochondrion
In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometers in diameter...
or chloroplast
Chloroplast
Chloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.Chloroplasts are green...
s. Several hundred of these enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s 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, intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
s or intein
Intein
An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions with a peptide bond. Inteins have also been called "protein introns"....
s. 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
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
. 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
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
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.
Homing endonucleases from the LAGLIDADG family
There are five families, or classes, of homing endonucleases. 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
I-CreI
I-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first such gene isolated...
(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
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
, 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).
Meganucleases as tools for genome engineering
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 engineeringGenome engineering
Genome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms....
.
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:
- Modifying the specificity of existing meganucleases by introducing a small number of variations to the amino acid sequence and then selecting the functional proteins on variations of the natural recognition site.
- A more radical option has been to exploit a property that plays an important role in meganucleases’ naturally high degree of diversification: the possibility of associating or fusing protein domains from different enzymes. This option makes it possible to develop chimeric meganucleases with a new recognition site composed of a half-site of meganuclease A and a half-site of protein B. By fusing the protein domains of I-DmoI and I-CreI, two chimeric meganucleases have been created using this method: E-Drel and DmoCre.
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. 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.
See also
- Homing endonucleaseHoming endonucleaseThe homing endonucleases are a type of restriction enzymes typically encoded by introns or inteins. They act on the cellular DNA of the cells that synthesize them, in the opposite alleles of the genes that encode them.- Origin and mechanism :...
- homologous recombinationHomologous recombinationHomologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
- I-CreII-CreII-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first such gene isolated...
- Protein engineeringProtein engineeringProtein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles....
- Protein designProtein designProtein design is the design of new protein molecules, either from scratch or by making calculated variations on a known structure. The use of rational design techniques for proteins is a major aspect of protein engineering....
- Genome engineeringGenome engineeringGenome engineering refers to the strategies and techniques developed in recent years for the targeted, specific modification of the genetic information – or genome – of living organisms....
- Genetic engineeringGenetic engineeringGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...