Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification. - CNRS - Centre national de la recherche scientifique Accéder directement au contenu
Article Dans Une Revue Proceedings of the National Academy of Sciences of the United States of America Année : 2008

Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification.

Fabio Cannata
  • Fonction : Auteur
Erika Brunet
Loïc Perrouault
  • Fonction : Auteur
Slimane Ait-Si-Ali
Jean-Paul Concordet
Carine Giovannangeli

Résumé

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.

Dates et versions

hal-00408029 , version 1 (28-07-2009)

Identifiants

Citer

Fabio Cannata, Erika Brunet, Loïc Perrouault, Victoria Roig, Slimane Ait-Si-Ali, et al.. Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification.. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105 (28), pp.9576-81. ⟨10.1073/pnas.0710433105⟩. ⟨hal-00408029⟩
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