Directed evolution of targeted recombinases for genome engineering

Abstract

Over the past several years, genome engineering has become an established component of basic research endeavors, and is emerging as a vital element of clinical research applications. Site-specific recombinases are one of the several tools that can facilitate genome modification by catalyzing rearrangements between specific DNA targets. Of particular interest are the small serine recombinases, which are modular in both form and function. This unique structure permits replacement of the native DNA-binding domain with designer targeting modules such as zinc fingers, TALEs, or catalytically inactivated Cas9, enabling modification of investigator-defined genomic loci. Importantly, the catalytic domain of these enzymes also contributes to target specificity, and can be reprogrammed to recognize custom sequences for genomic targeting. Here we describe the steps required to construct, select, and validate hybrid recombinase catalytic domains for targeted genome engineering.