Rational Engineering of Chimaeric Recombinases for Genomic Excision

Most current genome editing approaches being developed as potential therapeutic strategies utilize nuclease-based systems such as the TAL effector nucleases, Zinc-finger nucleases and CRISPR-Cas9. The mechanisms of action of these involve cleavage followed by homology-directed repair (HDR) or non-homologous end joining (NHEJ) at the cleavage site. However, off-target activities and fatal indel mutations characteristic of these approaches could have debilitating effects on the host cells and reduce the effectiveness of their application for precise gene therapy. Here, we present the rational engineering and in vitro characterization of Tal-effector recombinases (TALERs) capable of specific excision and religation on non-cognate sequences. These chimaeric recombinases comprise an N-terminal serine recombinase catalytic domain linked to a TAL effector C-terminal DNA-binding domain. We describe the essential TALER architecture, target site requirements and their in vitro recombination properties. As a proof of concept, we have engineered components of chimaeric recombinases to recognize and promote recombination at a highly conserved region within the HIV-1 long terminal repeats (LTR) flanking the proviral DNA at both ends. Each target sequence (one at each end of the provirus DNA) will be bound by two recombinase subunits, and then the two sites will be brought together, cleaved and recombined. As a result, the provirus is excised from the host genome (leaving behind a non-functional “scar” sequence). The requirement for interaction of two recombinase-bound sites, and the lack of necessity for any host cell-encoded factors should maximize the fidelity and efficiency of provirus removal. Our research to date has used a combination of random mutagenesis and rational engineering approaches to create a Tn3 resolvase-based recombinase catalytic domain capable of targeting the central part of our chosen HIV target sequence. We have also deduced the optimal structure and design of functional TAL effector chimaeric recombinases. Our current work is focused on assembling and characterizing the complete HIV-TAL effector recombinase and the validation of the engineered synthetic protein for in vivo genome editing.