Site-specific Recombinase Technology - Site-specific Recombinases: Classification, Properties and Dedicated Applications

Site-specific Recombinases: Classification, Properties and Dedicated Applications

Common genetic engineering strategies require a permanent modification of the target genome. To this end great sophistication has to be invested in the design of routes applied for the delivery of transgenes. Although for biotechnological purposes random integration is still common, it may result in unpredictable gene expression due to variable transgene copy numbers, lack of control about integration sites and associated mutations. The molecular requirements in the stem cell field are much more stringent. Here, homologous recombination (HR) can, in principle, provide specificity to the integration process, but for eukarytoes it is compromised by an extremely low efficiency. Although meganucleases, zinc-finger- and transcription activator-like effector nucleases (ZFNs and TALENs) are actual tools supporting HR, it was the availability of site-specific recombinases (SSRs) which triggered the rational construction of cell lines with predictable properties. Nowadays both technologies, HR and SSR can be combined in highly efficient "tag-and-exchange technologies".

Many site-specific recombination systems have been identified to perform these DNA rearrangements for a variety of purposes, but nearly all of these belong to either of two families, tyrosine recombinases (YR) and serine recombinases (SR), depending on their mechanism. These two families can mediate up to three types of DNA rearrangements (integration, excision/resolution, and inversion) along different reaction routes based on their origin and architecture.

The founding member of the YR family is the lambda integrase, encoded by bacteriophage λ, enabling the integration phage DNA into the bacterial genome. A common feature of this class is a conserved tyrosine nucleophile attacking the scissile DNA-phosphate to form a 3'-phosphotyrosine linkage. Early members of the SR family are closely related resolvase/invertases from the bacterial transposons Tn3 and γδ, which rely on a catalytic serine responsible for attacking the scissile phosphate to form a 5'-phosphoserine linkage. These undisputed facts, however, were compromised by a good deal of confusion at the time other members entered the scene, for instance the YR recombinases Cre and Flp (capable of integration, excision/resolution as well as inversion), which were nevertheless welcomed as new members of the "integrase family". The converse examples are PhiC31 and related SRs, which were originally introduced as resolvase/invertases although, in the absence of auxiliary factors, integration is their only function. Nowadays the standard activity of each enzyme determines its classification reserving the general term "recombinase" for family members which, per se, comprise all three routes, INT, RES and INV:

Figure 1: Tyr- and Ser-SSRs from prokaryotes (phages; grey) and eukaryotes (yeasts; brown); a comprehensive overview (including references) can be found in.

Our table extends the selection of the conventional SSR systems and groups these according to their performance. All enzymes listed in Fig. 1 recombine two target sites, which are either identical (subfamily A1) or distinct (phage-derived enzymes in A2, B1 and B2). Whereas for A1 these sites have individual designations ("FRT" in case of Flp-recombinase, "loxP" for Cre-recombinase), the terms "attP" and "attB" (attachment sites on the phage and bacterial part, respectively) are valid in the other cases. In case of subfamily A1 we have to deal with short (usually 34 bp-) sites consisting of two (near-)identical 13 bp arms (arrows) flanking an 8 bp spacer (the crossover region, indicated by red line doublets). Note that for Flp there is an alternative, 48 bp site available with three arms, each accommodating a Flp unit (a so called "protomer"). attP- and attB-sites follow similar architectural rules, but here the arms show only partial identity (indicated by the broken lines) and differ in both cases. These features account for relevant differences:

• recombination of two identical educt sites leads to product sites with the same composition, although they contain arms from both substrates; these conversions are reversible;
• in case of attP x attB recombination crossovers can only occur between these complementary partners in processes that lead to two different products (attP x attB → attR + attL) in an irreversible fashion.

In order to streamline this chapter the following implementations will be focused on two recombinases (Flp and Cre) and just one integrase (PhiC31) since their spectrum covers the tools which, at present, are mostly used for directed genome modifications. This will be done in the framework of the following overview (Fig. 2).