Cre can delete megabase regions and can mediate translocations between non-homologous chromosomes in mammalian genomes at remarkable efficiencies

Many applications have been found for site-specific recombinases (SSRs) in DNA and genome engineering. Cre recombinase from the coliphage P1 and FLP recombinase from the Saccharomyces cerevisiae 2 micron circle have become the most widely used SSRs (Branda and Dymecki, 2004; Glaser et al., 2005). Both belong to the class of tyrosine recombinases because they share a common reaction mechanism, involving nucleophilic attack on the phosphodiester backbone by a tyrosine hydroxyl group, to establish the covalent protein-DNA intermediate during recombination (Grindley et al., 2006). Cre and FLP mediate recombination between target sites, termed loxP and FRT, respectively, which are both 34 base pairs (bp) long and are based on 13-bp palindromes separated by 8-bp spacers. Recombination occurs between two loxP or FRT sites through the spacer regions. By deploying the target sites in different ways, a variety of different outcomes have been achieved. In DNA engineering, notable examples include the fluent conversion of λ phage inserts into plasmids (Elledge et al., 1991), removal of selectable genes (Zhang et al., 1998), linearization of bacterial artificial chromosomes (BACs) (Mullins et al., 1997) and biotin endlabeling (Buchholz and Bishop, 2001). In genome engineering, the most important applications include conditional mutagenesis (Gu et al., 1994; Kuhn et al., 1995; Logie and Stewart, 1995), gene expression switches (O’Gorman et al., 1991; Lakso et al., 1992; Angrand et al., 1998), lineage tracing (Awatramani et al., 2003), chromosomal engineering (Ramirez-Solis et al., 1995; Su et al., 2000; van der Weyden and Bradley, 2006), chromosomal translocations (Smith et al., 1995; Herault et al., 1998; Buchholz et al., 2000; Collins et al., 2000; Liu et al., 2002), recombinase-mediated cassette exchange (RMCE) (Schlake and Bode, 1994; Baer and Bode, 2001; Wallace et al., 2007), removal of selectable genes (Gu et al., 1993) and conversions of multipurpose alleles (Nagy et al., 1998; Testa et al., 2003; Testa et al., 2004; Schnutgen et al., 2005). The successes of Cre and FLP in genome engineering arise from their simplicity and efficiency. In terms of simplicity, both enzymes do not require any cofactors and their recombination target sites (RTs) are well defined and precisely understood (Senecoff et al., 1988; Lee and Saito, 1998). These RTs are small enough to be introduced very easily during DNA engineering, but large enough to minimize the problems associated with cryptic occurrence in eukaryotic genomes. The remarkable efficiency properties of Cre recombinase have emerged from many studies. For example, Cre can delete megabase regions and can mediate translocations between non-homologous chromosomes in mammalian genomes at remarkable efficiencies (Su et al., 2000; Wu et al., 2007). However, it was realized some time ago that FLP was less efficient than Cre, partly owing to an unsuitable optimum temperature for mammalian cells (Buchholz et al., 1996), which led to the application of molecular evolution to identify the thermostable variant called FLPe (Buchholz et al., 1998). However, FLPe is still less efficient than Cre, although a recent codon-optimized FLPe, termed FLPo, has bridged some of the remaining gap (Raymond and Soriano, 2007) (K.A., unpublished data). The weaker properties of FLP, as well as the merits in finding a third efficient recombinase for genome engineering, prompted searches for new tyrosine SSR tools (Araki et al., 1992; Ringrose et al., 1997; Christ et al., 2002). The search also encompassed RESOURCE ARTICLE