RETRACTED: 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 ≈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.

Chromosomal translocations induced at specified loci in human stem cells

“…Engineering of homing endonucleases to recognize an endogenous site in the genome, while challenging, has recently proved to be successful (36), providing an alternative approach to ZFNs for human genome modification. Synthetic chemical reagents have also recently been shown to lead to efficient DSB formation in the human genome (37,38). Our results, as well as advances in site-specific DSB technology, suggest that modification of the human genome for a variety of purposes, including translocation formation, will become increasingly possible and efficient in the near future.…”

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

“…However, utilization of OPEN requires an archive of pre-selected zinc-finger pools and E. coli selection (Maeder et al, 2008). Furthermore, due to the challenge of engineering the endonucleases, orthophenanthroline (OP, a DNA cleaving molecule) was conjugated with triplex-forming oligonucleotides (TFOs, sequence-specific binding capacity) to induce targeted DSBs and stimulate mutations at the target site in approximately 10% of treated human cells (Cannata et al, 2008). TFO conjugating to OP or other DNA cleaving molecules may provide a useful tool to induce targeted gene modification because triplex-forming sequences are frequent in mammalian genes.…”

Zinc-finger nucleases: a powerful tool for genetic engineering of animals

“…ZFNs have also been used to induce gene-targeted knock-ins in cells in the context of genome editing for gene therapy (Lombardo et al 2007;Moehle et al 2007;Perez et al 2008). Related technologies such as homing endonucleases utilize a different DNA cleavage mechanism, but the resulting DNA double strand break is resolved through the same mechanisms and engineered homing endonucleases Paques and Duchateau 2007;Stoddard et al 2008) or synthetic nucleases (Cannata et al 2008) can be applied in the same manner as ZFNs. The higher engineering barriers to generating novel homing endonucleases has so far limited their application to a few plant (Yang et al 2009) and human applications (Grizot et al 2009;Paques and Duchateau 2007), although recent progress should allow more widespread application as the engineering barriers to developing novel homing endonucleases are reduced (Arnould et al 2006;Ashworth et al 2006;Takeuchi et al 2009;Volna et al 2007).…”