Use of the IScel Endonuclease in Heterologous Systems

At the end of 1987,1 moved my laboratory from the CNRS at Gif-sur-Yvette to the Institut Pasteur in Paris. Given the exceptionally long recognition sequence of I-Scel, I was foreseeing a tool to cleave chromosomes, and possibly entire genomes, at a single predetermined site. Given the then emerging genome programs, especially the yeast sequencing project, YAC cloning and the first gene replacements in mammalian cells, this sounded to me like an attractive idea. One difficulty was the partial cleavage reactions produced when the universal code equivalent was expressed in heterologous systems. With Agnès Thierry, a young technician in my lab, I constructed a novel, totally synthetic gene for I-Scel, which has proven more efficient for expression, its codons being optimized for E. coli and yeast. This artificial gene has been distributed to nearly 150 laboratories in the world for academic research, and is also used for commercial production under agreement with Institut Pasteur. The sequence of the artificial gene is unpublished but can be obtained upon request. This gene has been successfully adapted with different promoters for in vivo expression in a very large variety of organisms including yeasts, fungi, insects, mammals, fishes, plants, ascidia, protozoa, bacteria, and viruses. A simple query of PubMed with the term "I-Scel" returns nearly 150 publications relating its use as a site-specific endonuclease for gene targeting and for studies on double-strand break repair, non-homologous end joining and other recombination mechanisms.

However, before these applications could start, a few technical details had to be worked out to demonstrate that I-Sce I could actually cleave only a single site in an entire eukaryotic genome. Yeast was instrumental for this demonstration (Thierry et al. 1991). To do this, artificial I-Scel sites were introduced at desired locations in its chromosomes by transformation with properly designed gene cassettes. Yeast chromosomes were then separated by the novel pulsed field gel electrophoresis technique. Treatment with I-Scel cleaved the yeast chromosome at the artificially inserted cognate site, leaving other chromosomes intact. This strategy has been very useful for the yeast sequencing program. Having inserted I-Scel sites at a large variety of chromosomal locations in different transformed clones, we could map the yeast genome at high resolution (Thierry and Dujon 1992; Tettelin et al. 1995,1998; Thierry et al. 1995). Similar experiments were done on YAC clones to map mouse genes (Colleaux et al. 1993; Rougeulle et al. 1994; Fairhead et al. 1995).

But the major interest was in the in vivo experiments. There was no indication that the protein, which naturally acts in mitochondria where it is synthesized, could ever enter the nucleus if synthesized on cytoplasmic ribosomes. For me, this was a good enough reason to try. The experiment was immediately successful. Induction of I-Scel by activation of an artificial promoter was generating specific double-strand breaks at desired locations in artificial yeast plasmids and in chromosomes (Plessis et al. 1992; Fairhead and Dujon 1993). This offered us a very powerful novel tool that we, and others, are now extensively using to study double-strand break repair mechanisms and other genome instability phenomena in yeast (Ricchetti et al. 1999,2003; Richard et al. 1999,2003), or to engineer the yeast genome (Fairhead et al. 1996,1998).

But yeast was not enough; the real challenge was higher eukaryotes. A young student in Jean-François Nicolas' lab, André Choulika, enthusiastically engaged in mouse cell experiments. Using specifically engineered retroviral vectors to insert I-Scel sites in the mouse genome, he rapidly demonstrated the power of this homing endonuclease to induce site-specific homologous recombination, opening the way for efficient gene replacement and targeting. Compared to controls, homologous recombination was stimulated almost 1000-fold (Choulika et al. 1994,1995). Many others have now done similar experiments in a variety of organisms (above). For myself, I was pleased to collaborate with Holger Puchta and Barbara Hohn in plant genetics (Puchta et al. 1993,1996) where the I-Scel homing endonuclease has great potential.

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