Engineering spacer specificity of the Cre/loxP system
Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing how Cre distinguishes between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking.  To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6,000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the flexibility of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations elucidated the mechanism behind this specificity switch. These findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications. Overall design: Characterizing  the activity of Cre and an evolved Cre variant, RecS3. In order to profile the activity, an oligo library of 6000 different target sites are assembled to an E. coli inducible expression vector. The vector carries the recombinase of interest, either Cre (Exp6) or RecS3 (Exp13). Once library plasmids are assembled, they are transformed to E. Coli and expression of the recombinase is induced over night. The subsequent vectors are then purified from the sample. In order to determine how many times each target was either edited (recombined) or not edited (non-recombined) we run a PCR over the target sites then send the amplicon for illumination deep sequencing. Each experiment is done in triplicates at different induction concentrations.