Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease |
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Authors: | Sylvestre Grizot Julianne Smith Fayza Daboussi Jesús Prieto Pilar Redondo Nekane Merino Maider Villate Séverine Thomas Laetitia Lemaire Guillermo Montoya Francisco J. Blanco Frédéric Paques Philippe Duchateau |
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Affiliation: | 1.Cellectis S.A., 2.Cellectis Genome Surgery, 102 Avenue Gustave Roussel, 93235 Romainville, France, 3.Structural Biology and Biocomputing Program, Spanish National Cancer Center (CNIO), Macromolecular Crystallography Group, Melchor Fdez. Almagro 3, 28029 Madrid and 4.Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Vizcaya, Edificio 800 48160, Derio, Spain |
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Abstract: | Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches. |
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