Identifying functional lox sequences: A genomic search and randomized libraries

Currently, homologous recombination is the standard for inserting exogenous DNA into host genomes, but the possibility exists that a site-specific recombinase system such as Cre/loxP could be manipulated as a more efficient means of delivering DNA into predetermined sites in a genome. Attempts have been made to find endogenous genomic lox sequences in a variety of organisms which could support efficient Cre/lox site-specific integration. So far, no highly effective genomic lox sequences have been found. In this study, two issues in the search for functionally efficient lox sites in genomes were addressed: the absence of a stable integration strategy when screening for genomic sites and the lack of knowledge about what constitutes a functional lox sequence. By applying existing knowledge to the in silico search for lox sites, this study shows that the possibility of finding such sites exists, but better understanding of what constitutes a functional site is needed. This study also evaluates what sequences can comprise the functional domains for the Cre recombinase through the testing, both in vivo and in vitro, of the first fully randomized libraries representing the two known loxP functional domains. Results from the spacer library show that identity between spacer pairs is the most important factor influencing function. Randomization of one arm of the loxP site revealed that the sequence of the randomized arm was not important for functional recombination in Cre expressing bacteria, and there are nucleotide preferences at the first three and last three positions of the randomized arm for efficient recombination.