Meganuclease

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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.

Contents

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.

Two main families

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.

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. 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.

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-palindromic (I-SceI).

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. Meganucleases were identified in the 1990s, and subsequent work has shown that they are particularly promising tools for genome engineering and gene editing, as they are able to efficiently induce homologous recombination, [2] generate mutations, [3] and alter reading frames. [4]

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 groups turned their attention to engineering new meganucleases that would target 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 have been working on gene editing since 1999 and have developed a collection of over 20,000 protein domains from the homodimeric meganuclease I-CreI as well as from other meganucleases scaffolds. [11] They can be combined to form functional chimeric tailor-made heterodimers for research laboratories and for industrial purposes.

Precision Biosciences, another 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. [12] In 2012 researchers at Bayer CropScience used DNE to incorporate a gene sequence into the DNA of cotton plants, targeting it precisely to a predetermined site. [13]

Additional applications

One recent advance in the use of meganucleases for genome engineering is the incorporation of the DNA binding domain from transcription activator-like (TAL) effectors into hybrid nucleases. These "megaTALs" combine the ease of engineering and high DNA binding specificity of a TAL effector with the high cleavage efficiency of meganucleases. [14] In addition, meganucleases have been fused to DNA end-processing enzymes in order to promote error-prone non-homologous end joining [15] and to increase the frequency of mutagenic events at a given locus. [16]

Probabilities

As stated in the opening paragraph, a meganuclease with an 18-base pair sequence would on average require a genome twenty times the size of the human genome to be found once by chance; the calculation is 418/3x109 = 22.9. However, very similar sequences are much more common, with frequency increasing quickly the more mismatches are permitted.

For example, a sequence that is identical in all but one base pair would occur by chance once every 417/18x3x109 = 0.32 human genome equivalents on average, or three times per human genome. A sequence that is identical in all but two base pairs would on average occur by chance once every 416/(18C2)x3x109 = 0.0094 human genome equivalents, or 107 times per human genome.

This is important because enzymes do not have perfect discrimination; a nuclease will still have some likelihood of acting even if the sequence does not match perfectly. So the activity of the nuclease on a sequence with one mismatch is less than the no-mismatch case, and activity is even less for two mismatches, but still not zero. Exclusion of these sequences, which are very similar but not identical, is still an important problem to be overcome in genome engineering.

Other considerations

DNA methylation and chromatin structure affect the efficacy of meganuclease digestion. [17] [18] A thorough consideration of the genetic and epigenetic context of a target sequence is therefore necessary for the practical application of these enzymes.

In December 2014, the USPTO issued patent 8,921,332 covering meganuclease-based genome editing in vitro. [19] This patent was licensed exclusively to Cellectis. [20]

See also

Related Research Articles

A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

The restriction modification system is found in bacteria and other prokaryotic organisms, and provides a defense against foreign DNA, such as that borne by bacteriophages.

Nuclease

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously affect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

Protein splicing The post-translational removal of peptide sequences from within a protein sequence

Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins on both sides. The splicing junction of the precursor protein is mainly a cysteine or a serine, which are amino acids containing a nucleophilic side chain. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP). Normally, splicing is associated only with pre-mRNA splicing. This precursor protein contains three segments—an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

Triple-stranded DNA DNA structure in which three oligonucleotides wind around each other and form a triple helix. In this structure, one strand binds to a B-form DNA double helix through Hoogsteen or reversed Hoogsteen hydrogen bonds

Triple-stranded DNA is a DNA structure in which three oligonucleotides wind around each other and form a triple helix. In triple-stranded DNA, the third strand binds to a B-form DNA double helix by forming Hoogsteen base pairs or reversed Hoogsteen hydrogen bonds.

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.

<i>Fok</i>I

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.

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 (I) such gene isolated from C. reinhardtii. Its gene resides in a group I intron in the 23S ribosomal RNA gene of the C. reinhardtii chloroplast, and I-CreI is only expressed when its mRNA is spliced from the primary transcript of the 23S gene. I-CreI enzyme, which functions as a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-CreI is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-CreI-containing intron encounters a 23S gene lacking the intron, I-CreI enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-CreI provides for its own propagation while conferring no benefit on its host, it is an example of selfish DNA.

Gene targeting

Gene targeting is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify individual base pairs. Gene targeting can be permanent or conditional. Conditions can be a specific time during development / life of the organism or limitation to a specific tissue, for example. Gene targeting requires the creation of a specific vector for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.

Homing endonuclease

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

APEX1

DNA-(apurinic or apyrimidinic site) lyase is an enzyme that in humans is encoded by the APEX1 gene.

Transcription activator-like effector

TALeffectors are proteins secreted by some β- and γ-proteobacteria. Most of these are Xanthomonads. Plant pathogenic Xanthomonas bacteria are especially known for TALEs, produced via their type III secretion system. These proteins can bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection. They recognize plant DNA sequences through a central repeat domain consisting of a variable number of ~34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. These proteins are interesting to researchers both for their role in disease of important crop species and the relative ease of retargeting them to bind new DNA sequences. Similar proteins can be found in the pathogenic bacterium Ralstonia solanacearum and Burkholderia rhizoxinica, as well as yet unidentified marine microorganisms. The term TALE-likes is used to refer to the putative protein family encompassing the TALEs and these related proteins.

Recombinant adeno-associated virus (rAAV) based genome engineering is a genome editing platform centered on the use of recombinant rAAV vectors that enables insertion, deletion or substitution of DNA sequences into the genomes of live mammalian cells. The technique builds on Mario Capecchi and Oliver Smithies' Nobel Prize–winning discovery that homologous recombination (HR), a natural hi-fidelity DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome-editing improves the efficiency of this technique to permit genome engineering in any pre-established and differentiated human cell line, which, in contrast to mouse ES cells, have low rates of HR.

Transcription activator-like effector nuclease

Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

Genome editing Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.

Genetic engineering techniques Methods used to change the DNA of organisms

Genetic engineering can be accomplished using multiple techniques. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism. The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them. Important advances included the discovery of restriction enzymes and DNA ligases and the development of polymerase chain reaction and sequencing.

References

  1. ↑ Stoddard, Barry L. (2006). "Homing endonuclease structure and function". Quarterly Reviews of Biophysics. 38 (1): 49–95. doi:10.1017/S0033583505004063. PMID   16336743. S2CID   27841011.
  2. ↑ Epinat, Jean-Charles; Arnould, Sylvain; Chames, Patrick; Rochaix, Pascal; Desfontaines, Dominique; Puzin, Clémence; Patin, Amélie; Zanghellini, Alexandre; Pâques, Frédéric (2003-06-01). "A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells". Nucleic Acids Research. 31 (11): 2952–2962. doi:10.1093/nar/gkg375. ISSN   1362-4962. PMC   156710 . PMID   12771221.
  3. ↑ Arnould, Sylvain; Perez, Christophe; Cabaniols, Jean-Pierre; Smith, Julianne; Gouble, Agnès; Grizot, Sylvestre; Epinat, Jean-Charles; Duclert, Aymeric; Duchateau, Philippe (2007-08-03). "Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells". Journal of Molecular Biology. 371 (1): 49–65. doi:10.1016/j.jmb.2007.04.079. ISSN   0022-2836. PMID   17561112.
  4. ↑ Chapdelaine, P.; Pichavant, C.; Rousseau, J.; Pâques, F.; Tremblay, J. P. (2010-07-01). "Meganucleases can restore the reading frame of a mutated dystrophin". Gene Therapy. 17 (7): 846–858. doi: 10.1038/gt.2010.26 . ISSN   1476-5462. PMID   20393509.
  5. ↑ Seligman, L. M.; 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 (17): 3870–9. doi:10.1093/nar/gkf495. PMC   137417 . PMID   12202772.
  6. ↑ Sussman, Django; Chadsey, Meg; Fauce, Steve; Engel, Alex; Bruett, Anna; Monnat, Ray; Stoddard, Barry L.; Seligman, Lenny M. (2004). "Isolation and Characterization of New Homing Endonuclease Specificities at Individual Target Site Positions". Journal of Molecular Biology. 342 (1): 31–41. doi:10.1016/j.jmb.2004.07.031. PMID   15313605.
  7. ↑ Rosen, L. E.; Morrison, H. A.; Masri, S.; Brown, M. J.; Springstubb, B.; Sussman, D.; Stoddard, B. L.; Seligman, L. M. (2006). "Homing endonuclease I-CreI derivatives with novel DNA target specificities". Nucleic Acids Research. 34 (17): 4791–800. doi:10.1093/nar/gkl645. PMC   1635285 . PMID   16971456.
  8. ↑ Arnould, Sylvain; Chames, Patrick; Perez, Christophe; Lacroix, Emmanuel; Duclert, Aymeric; Epinat, Jean-Charles; Stricher, François; Petit, Anne-Sophie; Patin, Amélie (2006). "Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets". Journal of Molecular Biology. 355 (3): 443–58. doi:10.1016/j.jmb.2005.10.065. PMID   16310802.
  9. ↑ Smith, J.; Grizot, S.; Arnould, S.; Duclert, A.; Epinat, J.-C.; Chames, P.; Prieto, J.; Redondo, P.; Blanco, F. J. (2006). "A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences". Nucleic Acids Research. 34 (22): e149. doi:10.1093/nar/gkl720. PMC   1702487 . PMID   17130168.
  10. ↑ Chevalier, Brett S.; Kortemme, Tanja; Chadsey, Meggen S.; Baker, David; Monnat, Raymond J.; Stoddard, Barry L. (2002). "Design, Activity, and Structure of a Highly Specific Artificial Endonuclease". Molecular Cell. 10 (4): 895–905. doi: 10.1016/S1097-2765(02)00690-1 . PMID   12419232.
  11. ↑ Grizot, S.; Epinat, J. C.; Thomas, S.; Duclert, A.; Rolland, S.; Paques, F.; Duchateau, P. (2009). "Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds". Nucleic Acids Research. 38 (6): 2006–18. doi:10.1093/nar/gkp1171. PMC   2847234 . PMID   20026587.
  12. ↑ Gao, Huirong; Smith, Jeff; Yang, Meizhu; Jones, Spencer; Djukanovic, Vesna; Nicholson, Michael G.; West, Ande; Bidney, Dennis; Falco, S. Carl (2010). "Heritable targeted mutagenesis in maize using a designed endonuclease". The Plant Journal. 61 (1): 176–87. doi: 10.1111/j.1365-313X.2009.04041.x . PMID   19811621.
  13. ↑ http://www.research.bayer.com/en/straight-into-the-cotton-genome.aspx%5B%5D
  14. ↑ Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L.; Certo, Michael T. (2014-02-01). "megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering". Nucleic Acids Research. 42 (4): 2591–2601. doi:10.1093/nar/gkt1224. ISSN   1362-4962. PMC   3936731 . PMID   24285304.
  15. ↑ Certo, Michael T; Gwiazda, Kamila S; Kuhar, Ryan; Sather, Blythe; Curinga, Gabrielle; Mandt, Tyler; Brault, Michelle; Lambert, Abigail R; Baxter, Sarah K (2012-10-01). "Coupling endonucleases with DNA end-processing enzymes to drive gene disruption". Nature Methods. 9 (10): 973–975. doi:10.1038/nmeth.2177. ISSN   1548-7091. PMC   3602999 . PMID   22941364.
  16. ↑ Delacôte, Fabien; Perez, Christophe; Guyot, Valérie; Duhamel, Marianne; Rochon, Christelle; Ollivier, Nathalie; Macmaster, Rachel; Silva, George H.; Pâques, Frédéric (2013-01-01). "High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes". PLOS ONE. 8 (1): e53217. Bibcode:2013PLoSO...853217D. doi: 10.1371/journal.pone.0053217 . ISSN   1932-6203. PMC   3554739 . PMID   23359797.
  17. ↑ Valton, Julien; Daboussi, Fayza; Leduc, Sophie; Molina, Rafael; Redondo, Pilar; Macmaster, Rachel; Montoya, Guillermo; Duchateau, Philippe (2012-08-31). "5′-Cytosine-Phosphoguanine (CpG) Methylation Impacts the Activity of Natural and Engineered Meganucleases". The Journal of Biological Chemistry. 287 (36): 30139–30150. doi: 10.1074/jbc.M112.379966 . ISSN   0021-9258. PMC   3436367 . PMID   22740697.
  18. ↑ Daboussi, Fayza; Zaslavskiy, Mikhail; Poirot, Laurent; Loperfido, Mariana; Gouble, Agnès; Guyot, Valerie; Leduc, Sophie; Galetto, Roman; Grizot, Sylvestre (2012-03-29). "Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases". Nucleic Acids Research. 40 (13): 6367–6379. doi:10.1093/nar/gks268. ISSN   0305-1048. PMC   3401453 . PMID   22467209.
  19. ↑ "US Patent for Chromosomal modification involving the induction of double-stranded DNA cleavage and homologous recombination at the cleavage site Patent (Patent # 8,921,332 issued December 30, 2014) - Justia Patents Search". patents.justia.com. Retrieved 2016-02-19.
  20. ↑ "Cellectis announces the issuance by the USPTO of a patent covering a seminal nuclease-based "gene editing" method | cellectis". www.cellectis.com. Archived from the original on 2016-03-02. Retrieved 2016-02-19.
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