title: Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification.
abstract: The inefficiency of gene modification by homologous recombination can be overcome by the introduction of a double-strand break (DSB) in the target. Engineering the endonucleases needed, however, remains a challenging task that limits widespread application of nuclease-driven gene modification. We report here that conjugates of orthophenanthroline (OP), a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences, are synthetic nucleases efficient at stimulating targeted genome modification. We show that in cultured cells, OP-TFO conjugates induce targeted DSBs. An OP-TFO with a unique target was highly efficient, and mutations at the target site were found in approximately 10% of treated cells, including small deletions most likely introduced during DSB repair by nonhomologous end joining. Importantly, we found that when homologous donor DNA was cotransfected, targeted gene modification took place in >1.5% of treated cells. Because triplex-forming sequences are frequent in human and mouse genes, OP-TFO conjugates therefore constitute an important class of site-specific nucleases for targeted gene modification. Harnessing DNA-damaging molecules to predetermined genomic sites, as achieved here, should also provide inroads into mechanisms of DNA repair and cancer.
authors.affiliation: Laboratoire de Biophysique, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5153 USM 503, and Institut National de la Santé et de la Recherche Médicale U565, Muséum National d'Histoire Naturelle, 43 Rue Cuvier, 75005 Paris, France.
authors.forename: Fabio
authors.initials: F
authors.lastname: Cannata
authors.forename: Erika
authors.initials: E
authors.lastname: Brunet
authors.forename: Loïc
authors.initials: L
authors.lastname: Perrouault
authors.forename: Victoria
authors.initials: V
authors.lastname: Roig
authors.forename: Slimane
authors.initials: S
authors.lastname: Ait-Si-Ali
authors.forename: Ulysse
authors.initials: U
authors.lastname: Asseline
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Concordet
authors.forename: Carine
authors.initials: C
authors.lastname: Giovannangeli
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004720:Endonucleases; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D006801:Humans; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D010618:Phenanthrolines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D009841:Oligonucleotides; D010618:Phenanthrolines; D004720:Endonucleases; C025205:1,10-phenanthroline