Meganuclease

Meganuclease

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.

Contents

Two main families

Generalized homing mechanisms for mobile group I introns
Generalized homing mechanisms for mobile group I introns

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.

Homing endonucleases from the LAGLIDADG family

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).

Meganucleases as tools for genome engineering

The DmoCre protein
The DmoCre protein; Extract from the article Grizot S, Epinat JC, Thomas S, Duclert A, Rolland S, Pâques F, and Duchateau P. (2010) Generation of redesigned homing endonucleases comprising DNA-binding domain derived from two different scaffolds. Nucleic Acids Research. 38(6): 2006–2018. © The Author(s) 2009. Published by Oxford University Press.

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:

  • 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[2][3][4].
  • 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[5][6]. 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[7].

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].

See also

Astronaut-EVA.jpg Technology and applied sciences portal

References

Explanatory video about meganucleases, by Cellectis

  1. ^ BL. (2006) Homing endonuclease structure and function. Quarterly Reviews of Biophysics 38, 1, pp. 49–95.
  2. ^ LM, Chisholm KM, Chevalier BS, Chadsey MS, Edwards ST, Savage JH, Veillet AL. (2002) Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research. 30:3870-3879.
  3. ^ D, Chadsey M, Fauce S, Engel A, Bruett A, Monnat R, Stoddard BL, Seligman LM. (2004) Isolation and Characterization of New Homing Endonuclease Specificities at Individual Target Site Positions. Journal of Molecular Biology. 342:31-41.
  4. ^ Rosen LE, Morrison HA, Masri S, Brown MJ, Springstubb B, Sussman D, Stoddard BL, Seligman LM. (2006) Homing endonuclease I-CreI derivatives with novel DNA target specificities. Nucleic Acids Research. 34:4791-4800.
  5. ^ S, Chames P, Perez C, Lacroix E, Duclert A, Epinat JC, Stricher F, Petit AS, Patin A, Guillier S, Rolland S, Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, Duchateau P, Pâques F. (2006) Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets. Journal of Molecular Biology. 355:443-458.
  6. ^ J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, Prieto J, Redondo P, Blanco FJ, Bravo J, Montoya G, Pâques F, Duchateau P. (2006) A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Research. 34(22):e149.
  7. ^ BS, Kortemme T, Chadsey MS, Baker D, Monnat RJ, ad Stoddard BL. (2002) Design, activity, and structure of a highly specific artificial endonuclease. Mol Cell. 10(4):895-905.
  8. ^ S, Epinat JC, Thomas S, Duclert A, Rolland S, Pâques F, and Duchateau P. (2010) Generation of redesigned homing endonuceases comprising DNA-binding domain derived from two different scaffolds. Nucleic Acids Research. 38(6): 2006–2018.
  9. ^ Huirong Gao; James Smith; Maizhu Yang; Spencer Jones; Jessica Stagg; Vesna Djukanovic; Mike Nicholson; Ande West; Dennis Bidney; Carl Falco; Derek Jantz; L. Alexander Lyznik, Heritable Targeted Mutagenesis in Maize Using a Dedicated Meganuclease, The Plant Journal 61 (1): 176-87, 2009

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