2008/01/01 by Sylvestre Grizot, Julianne Smith, Fayza Daboussi +11 · 6 citations
Biochemistry, Genetics and Molecular Biology · Social Sciences · #Advanced biosensing and bioanalysis techniques #CRISPR and Genetic Engineering #Digitalization, Law, and Regulation #Intellectual Property Rights and Media #Law and Political Science #RNA Interference and Gene Delivery
paper · pdf · doi:10.1093/nar/gkp548
openalex publication_date 2008/01/01 · openalex created_date 2016/06/24 · openalex updated_date 2026/04/28
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.