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 cano nical 34 bp loxP site, but can recombine with each other. By placing d ifferent 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 endo genous 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 'spa cer' region of the targeted chromosomal element, Moreover, in cells ex pressing a catalytically active Cre recombinase, naturally occurring l ex-like sequences can exhibit almost 20% mitotic recombination, Thus, in the same cell, heterospecific lex sites can be used independently a t multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulat ions catalyzed by the Cre recombinase.