Induction of Homologous Recombination in Mammalian Chromosomes by Using the I-SceI System of Saccharomyces cerevisiae

Abstract

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous with regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.Homologous recombination (HR) between chromosomal and exogenous DNAs is at the basis of methods for introducing genetic changes into the genome (5,20). Parameters of the recombination mechanism have been determined by studying plasmid sequences introduced into cells (1,4,10,11) and in an in vitro system (8). HR is inefficient in mammalian cells but is promoted by double-strand breaks in DNA.So far, it has not been possible to cleave a specific chromosomal target efficiently, thus limiting our understanding of recombination and its exploitation. Among endonucleases, the Saccharomyces cerevisiae mitochondrial endonuclease I-SceI (6) has characteristics which can be exploited as a tool for cleaving a specific chromosomal target and, therefore, manipulating the chromosome in living organisms. I-SceI protein is an endonuclease responsible for intron homing in yeast mitochondria, a nonreciprocal mechanism by which a predetermined sequence becomes inserted at a predetermined site. It has been established that the I-SceI endonuclease can induce recombination in yeast nuclei (16) and can enhance extrachromosomal recombination in COS-1 cells (17) by initiating a double-strand break. The recognition site of the I-SceI endonuclease is 18 bp long; therefore, I-SceI recognition sequences are very rare in genomic DNA (22). In addition, as the I-SceI protein is not a recombinase, its potential for chromosome engineering is larger than that of systems with target site requirements on both host and donor molecules (9).We demonstrate here that the yeast I-SceI endonuclease can efficiently induce double-strand breaks in a chromosomal target in mammalian cells and that the breaks can be repaired with a donor molecule homologous with the regions flanking the break. The enzyme catalyzes recombination at a high efficiency. This demonstrates that recombination between chromosomal DNA and exogenous DNA can occur in mammalian c...

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

“…Expression of I-SceI stimulated HR by 155-fold, which is in good agreement with previously published results 4,8,30 28-31 , corresponding to 11% of transfected cells undergoing HR between the target locus and the repair plasmid. After transfection of the original ZFN expression vectors (WT-WT), the number of transfected cells after seven days was too low to assess the frequency of HR, probably due to high toxicity.…”

“…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.…”

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.…”

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.…”