Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

Abstract

To maintain genomic integrity, double-strand breaks (DSBs) in chromosomal DNA must be repaired. In mammalian systems, the analysis of the repair of chromosomal DSBs has been limited by the inability to introduce well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression system for a rare-cutting endonuclease, I-Scel. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-Scel have been integrated into the mouse genome in two tandem neomycin phosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having at least one cleavage event and, of these, more than 70% have undergone cleavage at both I-Scel sites. Cleavage of both sites in a fraction of clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechanisms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparently recombinogenic, stimulating gene targeting of a homologous fragment by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent approximately 10% of cells transfected with the I-Scel expression vector. Gene targeted clones are of two major types, those that occur by two-sided homologous recombination with the homologous fragment and those that occur by one-sided homologous recombination. Our results are expected to impact a number of areas in the study of

Gene targeting is enhanced in human cells overexpressing hRAD51

“…[7][8][9][10] Substantial increases have been obtained by engineering double-strand breaks (DSBs) in the target locus, but this approach is not currently applicable to single-step targeting strategies. [11][12][13][14] Recent approaches to the problem have included the use of short fragments of DNA, 15 DNA/RNA oligonucleotides (reviewed in Ref. 16), modified selection conditions 17 and adeno-associated virus vectors 18 and these have generated some dramatically improved targeting efficiencies.…”

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…Several recent studies have reported gene-targeting frequencies of 1-18% HR events per 5 mammalian cell when the targeted DSB was introduced by natural or artificial endonucleases [2][3][4][5][6][7][8] . The ability to generate precise modifications of the genome -knockouts, mutations or corrections -within 1 in 100 to 1 in 5 cells presents this methodology as a potentially powerful tool for genetic studies, biotechnology and gene therapy.…”

Meganucleases can restore the reading frame of a mutated dystrophin

“…24 They can induce site-specific DSBs, and thereby stimulate homologous recombination of up to 10 000-fold in cultured cells. 25,26 MGNs were used to induce homologous recombination in a variety of cell types and organisms, including mammalian cells, mice, plants, Drosophila, Escherichia coli and trypanosome. 27,28 MGN-induced DSB can also be repaired by non-homologous end-joining (NHEJ), an error-prone process, which frequently results in micro-insertions or micro-deletions (indels) at the site of the break.…”