Development of tools for directed evolution of high-specificity, high-efficiency recombinases

PROJECT SUMMARY/ABSTRACT Precision genome editing presents opportunities for treatment of disease and for use in biotechnology. Tyrosine family site-specific DNA recombinases (Y-SSRs), such as Cre, are uniquely positioned as chemical biology tools for genetic engineering because they are capable of excision, integration, and inversion of DNA sequences without requiring exogenous co-factors. A major focus in tyrosine recombinase research is the retargeting of these enzymes to new DNA sites, primarily using substrate-linked directed evolution (SLiDE). Although successful in many regards, SLiDE has two substantial drawbacks that limit its use: limited capacity for negative selection can lead to promiscuous recombinases, and lack of selection tunability limits the ability to select for highly active recombinases. Off-target activities could have serious negative consequences in the human genome, limiting the use of evolved recombinases as probes or eventual therapeutics against human disease. Rigorous characterization of the activity of evolved recombinases towards off-target sites and the development of methods to lower or eliminate these off-target activities is a high-priority enabling technology. Likewise, development of a directed evolution scheme that can select for high recombination efficiency would enable the production of recombinase libraries with higher average efficiency in less time. Here, we aim to address both specificity and efficiency of recombination during evolution by developing novel SLiDE methodology. First, to combat promiscuity in evolved recombinases, we will develop a SLiDE method that leverages simultaneous positive selection at a desired target site and negative selection against a library of off- target sequences. We will use this method to evolve novel recombinases for activity against loxHTLV, the target of the previously-engineered RecHTLV. The activity of novel recombinases with the off-target library will then be quantified and compared to that of wild-type Cre and RecHTLV to observe differences in their promiscuity profiles. Second, to enable selection for higher efficiency recombinases, we will establish a bacterial system by introducing toxic “kill switches” into evolution vectors that are temperature controlled. Recombinase-expressing E. coli that have been transformed with these vectors will select for enzymes that can excise the toxic gene from all plasmid copies before timed activation of the kill switch. We will validate this system with existing recombinases and then evolve a new recombinase for activity on loxHTLV to compare to the activity and evolutionary timeline of the previously evolved RecHTLV. We hypothesize that this novel selection for recombination efficiency will rapidly produce an enzyme with improved efficiency in fewer rounds of evolution. Development of these selection systems will accelerate the development of specific, efficient evolved recombinases, streamlining the timeline for development of novel tools and potential therapeutics.