Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome

Variant lex sites having an altered spacer region (heterospecific lex sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lex sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated, Such heterospecific lex sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lex-like sequences that naturally occur in the genome, Specific targeting occurs only with a DNA vector carrying a heterospecific lex site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element, Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lex-like sequences can exhibit almost 20% mitotic recombination, Thus, in the same cell, heterospecific lex sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.