Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

Abstract

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.

Site-specific recombinases: molecular machines for the Genetic Revolution

“…First, SSRs including Cre and FLP have been subjected to directed evolution, selecting for increased activity on pseudosites having some resemblance to the natural recombination site [131][132][133][134]. Notable successes of this approach include the targeting of specific sequences in the human genome [135,136] and the HIV long terminal repeat (LTR) [137][138][139] (recombination between the identical sequences in the LTRs at each end of the HIV proviral genome could excise the viral DNA and thus form the basis of a therapy). This directed evolution strategy tends, however, to lead to the selection of variants with broadened rather than switched specificity.…”

Advances in New Technology for Targeted Modification of Plant Genomes

“…In 1998 Buchholz performed error-prone PCR on the Flp gene, inducing a series of mutations that increased the enzyme's thermo-stability and relative rates of activity. Second generation modifi cations included mutagenesis for increased activity (Buchholz et al 1996 ), recognition site alteration (Abi-Ghanem et al 2013 ;Keravala et al 2008 ;Sclimenti et al 2001 ), codon optimization for increased rates of translation and thus higher cellular concentration (Blechl et al 2012 ;Moon et al 2011 ;Raymond and Soriano 2007 ), and addition of nuclear localization signals (NLS) to increase effective concentrations of the recombinase in the nucleus (Andreas et al 2002 ;Blechl et al 2012 ). While these modifi cations have been effective in enhancing activity or altering binding site recognition, they did not fundamentally change the structure of the recombinase or allow it to recognize offsite targets.…”

6. Themenbereich somatische Gentherapie: Translationale und klinische Forschung

“…Designernukleasen) 40 zur Verfügung, welche es erlauben, das Zielzellgenom hochspezifisch zu editieren (Übersicht: Händel/Cathomen, 2011). Alternativ zu den Nukleasen werden zudem natürlich vorkommende Rekombinasen so modifiziert, dass sie neue Zielsequenzen erkennen (Sarkar et al, 2007;Abi-Ghanem et al, 2013).…”