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| Molecular Therapy 2008 | DOI:10.1038/mt.2008.118 |
| Methods Mol Biol. 2008;435:31-45 | DOI:10.1007/978-1-59745-232-8_3 |
| Nucleic Acids Res. published online on February 14, 2008 | DOI:10.1093/nar/gkn059 |
| Journal of Biological Chemistry published as Manuscript M706323200 on November 12, 2007 | DOI:10.1074/jbc.M706323200 |
| Journal of Molecular Biology published online on May 10, 2007 | DOI:10.1016/j.jmb.2007.04.079 |
| Nucleic Acids Res. published online on April 22, 2007 | DOI: 10.1093/nar/gkm183 |
A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
| Nucleic Acids Res. 2006;34(22):e149 | DOI: 10.1093/nar/gkl720 |
Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, Prieto J1, Redondo P1, Blanco FJ1, Bravo J1, Montoya G1, Pâques F*, Duchateau P.
CELLECTIS S.A., 102 route de Noisy, 93235 Romainville, France.
1Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO) C/ Melchor Fdez Almagro, 28029 Madrid, Spain
*To whom correspondence should be addressed. Tel: +33 1 41 83 99 00; Fax: +33 1 41 83 99 03; Email: paques@cellectis.com
| Abstract |
Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.
Full Text Article at http://nar.oxfordjournals.org
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Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break.
| J Gene Med 2006; 8: 616-622. | DOI: 10.1002/jgm.879 |
Agnès Gouble1 ♣, Julianne Smith1 ♣, Sylvia Bruneau1, Christophe Perez1, Valérie Guyot1, Jean-Pierre Cabaniols1, Sophie Leduc 1, Laurence Fiette2, Patrick Avé2, Béatrice Micheau1, Philippe Duchateau1 *, Frédéric Pâques1
1 CELLECTIS S.A., 102 route de Noisy, 93235 Romainville, France
2 U.R.E. Histotechnologie et Pathologie, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France
♣ These authors contributed equally to this work
* Correspondence to Philippe Duchateau, CELLECTIS S.A., 102 route de Noisy, 93235 Romainville, France (Philippe.Duchateau@cellectis.com)
Funded by : l'Agence Française de l'Innovation (ANVAR)
Keywords : homologous recombination, meganuclease, gene correction, single-strand annealing
| Abstract |
Background
Sequence-specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double-strand break (DSB)-induced HR.
Methods
In order to investigate DSB-induced HR in toto, we have designed transgenic mouse lines carrying a LagoZ gene interrupted by one I-SceI cleavage site surrounded by two direct repeats. The LagoZ gene can be rescued upon cleavage by I-SceI and HR between the two repeats in a process called single-strand annealing. β-Galactosidase activity is monitored in liver after tail vein injection of adenovirus expressing the meganuclease I-SceI.
Results
In toto staining revealed a strong dotted pattern in all animals injected with adenovirus expressing I-SceI. In contrast, no staining could be detected in the control. β-Galactosidase activity in liver extract, tissue section staining, and PCR analysis confirmed the presence of the recombined LagoZ gene.
Conclusions
We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy. Copyright © 2006 John Wiley & Sons, Ltd.
Full Text Article at Wiley InterScience |
In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination.
Nucleic Acids Res. 2005 33(20):e178
Patrick Chames, Jean-Charles Epinat, Sophie Guillier, Amélie Patin, Emmanuel Lacroix and Frédéric Pâques
CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France
| Abstract |
Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering.
Full Text Article at nar.oxfordjournals.org |
Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets.
J Mol Biol. 2006 Jan 20;355(3):443-58. Epub 2005 Nov 15.
Sylvain Arnould, Patrick Chames, Christophe Perez, Emmanuel Lacroix, Aymeric Duclert, Jean-Charles Epinat, François Stricher, Anne-Sophie Petit, Amélie Patin, Sophie Guillier, Sandra Rolland, Jesús Prieto, Francisco J. Blanco, Jerónimo Bravo, Guillermo Montoya, Luis Serrano, Philippe Duchateau and Frédéric Pâques.
CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France
Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncologica, C/ Melchor Fdez Almagro, 28029 Madrid, Spain.
European Molecular Biology Laboratory, Meyerhofstrasse D-62117 Heidelberg, Germany.
| Abstract |
The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.
Full Text Article at www.sciencedirect.com |
Factors affecting double-strand break-induced homologous recombination in mammalian cells.
BioTechniques® July 2005 ; Volume 39, Number 1: pp 109-115
Christophe Perez, Valérie Guyot, Jean-Pierre Cabaniols, Agnès Gouble, Beatrice Micheaux, Julie Smith, Sophie Leduc, Frédéric Pâques, and Philippe Duchateau
CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France.
| Abstract |
Double-strand break (DSB)-induced homologous recombination (HR) of direct repeats is a powerful means to achieve gene excision, a critical step in genome engineering. In this report we have used an extrachromosomal reporter system to monitor the impact of different parameters on meganuclease-induced HR in CHO-K1 cells. We found that repeat homology length is critical. Virtually no HR could be detected with a 15-bp duplication, while, with repeats larger than 400 bp, recombination efficiency became less dependent on homology length. The presence of an intervening sequence between the duplications dramatically impairs HR, independent of the cleavage position; by 3 kb of insertion, HR is virtually undetectable. Efficient HR can be restored by positioning cleavage sites at both ends of the intervening sequence, allowing a constant level of excision with up to 10 kb of intervening sequences. Using similar constructs, 2.8-kb inserts could be efficiently removed from several chromosomal loci, illustrating the wide potential of this technology. These results fit current models of direct repeat recombination and identify DSB-induced HR as a powerful tool for gene excision.
Full Text Article at www.biotechniques.com |
A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.
Nucleic Acids Res. 2003 Jun 1;31(11):2952-62
Epinat JC, Arnould S, Chames P, Rochaix P, Desfontaines D, Puzin C, Patin A, Zanghellini A, Paques F, Lacroix E.
CELLECTIS S.A., 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
| Abstract |
Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.
Full Text Article at nar.oupjournals.org |
I-SceI meganuclease mediates highly efficient transgenesis in fish.
Mech Dev. 2002 Oct;118(1-2):91-8
Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS.
INRA Junior Group, UPR2197, Institut de Neurobiologie A. Fessard, CNRS, Avenue de la Terrasse, 91 198 Gif-Sur-Yvette, France.
| Abstract |
| The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection. |
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