Genome-wide Mapping of Off-Target Events in Single-Stranded Oligodeoxynucleotide-Mediated Gene Repair Experiments.

Short single-stranded oligodeoxynucleotides are versatile molecular tools used in different applications. They enable gene repair and genome editing, and they are central to the antisense technology. Because the usability of single-stranded oligodeoxynucleotides depends on their efficiencies, as well as their specificities, analyzing their genotoxic off-target activities is important.

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary.

Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications.

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODN-mediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation.

Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break.

A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction.

Physical incorporation of a single-stranded oligodeoxynucleotide during targeted repair of a human chromosomal locus.

Background: Targeted gene repair is an attractive method to correct point-mutated genes at their natural chromosomal sites, but it is still rather inefficient. As revealed by earlier studies, successful gene correction requires a productive interaction of the repair molecule with the target locus. The work here set out to investigate whether DNA repair, e.

Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene.

Background: A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts).