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{{Short description|Genome engineering tools}}
{{technical|date=May 2019}}
'''Site-specific recombinase technologies''' are [[genome engineering]] tools that depend on [[Recombinase|recombinase enzymes]] to replace targeted sections of DNA.
== History ==
In the late 1980s gene targeting in murine [[embryonic stem cell
==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 eukaryotes 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".<ref>{{cite journal |doi=10.1016/S1534-5807(03)00399-X |title=Talking about a RevolutionThe Impact of Site-Specific Recombinases on Genetic Analyses in Mice |year=2004 |last1=Branda |first1=Catherine S. |last2=Dymecki |first2=Susan M. |authorlink2=Susan Dymecki|journal=Developmental Cell |volume=6 |pages=7–28 |pmid=14723844 |issue=1|doi-access=free }}</ref>
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 [[site-specific recombination|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.<ref name= "nern">{{cite journal |doi=10.1073/pnas.1111704108 |bibcode=2011PNAS..10814198N |title=Multiple new site-specific recombinases for use in manipulating animal genomes |year=2011 |last1=Nern |first1=A. |last2=Pfeiffer |first2=B. D. |last3=Svoboda|author3-link=Karel Svoboda (scientist) |first3=K. |last4=Rubin |first4=G. M. |journal=Proceedings of the National Academy of Sciences |volume=108 |issue=34 |pages=14198–203 |pmid=21831835 |pmc=3161616|doi-access=free }}</ref>
[[File:Classification_of_site-specific_recombinases_according_to_mechanism.png|thumb|755x755px|'''Tyr- and Ser-SSRs from prokaryotes''' (phages; grey) '''and eukaryotes''' (yeasts; brown); a comprehensive overview (including references) can be found in.<ref name="turan">{{cite journal|last2=Bode|first2=J.|year=2011|title=Site-specific recombinases: From tag-and-target- to tag-and-exchange-based genomic modifications|journal=The FASEB Journal|volume=25|issue=12|pages=4088–107|doi=10.1096/fj.11-186940|pmid=21891781|last1=Turan|first1=S.|doi-access=free |s2cid=7075677}}</ref>]]
The founding member of the YR family is the [[lambda integrase]], encoded by [[Bacteriophage| bacteriophage λ]], enabling the integration of 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 [[wiktionary:resolvase|resolvase]] / [[DNA invertase]]s 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 recombinase|Cre]] and [[FLP-FRT recombination|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:
Our table extends the selection of the conventional SSR systems and groups these according to their performance. All of
* 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;
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GOI, "gene of interest"; [+/-], a positive-negative selection marker such as the hygtk-fusion gene. Note that interaction of two identical substrate sites (loxP x loxP or FRT x FRT) leads to products of the same composition, whereas recombination of two non-identical educts leads to two different hybrid sites (attP x attB → attR + attL)]]
== Reaction routes
The mode integration/resolution and inversion (INT/RES and INV) depend on the orientation of recombinase target sites (RTS), among these pairs of ''att''P and ''att''B. Section C indicates, in a streamlined fashion, the way [[recombinase-mediated cassette exchange]] (RMCE) can be reached by synchronous double-reciprocal crossovers (rather than integration, followed by resolution).<ref>{{cite journal |doi=10.1093/nar/21.4.969 |title=Activity of yeast FLP recombinase in maize and rice protoplasts |year=1993 |last1=Lyznik |first1=Leszek A. |last2=Mitchell |first2=Jon C. |last3=Hirayama |first3=Lynne |last4=Hodges |first4=Thomas K. |journal=Nucleic Acids Research |volume=21 |issue=4 |pages=969–75 |pmid=8451196 |pmc=309231}}</ref><ref>{{cite journal |doi=10.1093/nar/gnf114 |title=Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases |year=2002 |last1=Lauth |first1=M. |journal=Nucleic Acids Research |volume=30 |issue=21 |pages=115e |pmid=12409474 |last2=Spreafico |first2=F |last3=Dethleffsen |first3=K |last4=Meyer |first4=M |pmc=135837}}</ref>
Tyr-Recombinases are reversible, while the Ser-Integrase is unidirectional. Of note is the way reversible Flp (a Tyr recombinase) integration/resolution is modulated by 48 bp (in place of 34 bp minimal) ''FRT'' versions: the extra 13 bp arm serves as a Flp "landing path" contributing to the formation of the synaptic complex, both in the context of Flp-INT and Flp-RMCE functions (see the respective equilibrium situations). While it is barely possible to prevent the (entropy-driven) reversion of integration in section A for Cre and hard to achieve for Flp, RMCE can be completed if the donor plasmid is provided at an excess due to the bimolecular character of both the forward- and the reverse reaction. Posing both ''FRT'' sites in an inverse manner will lead to an equilibrium of both orientations for the insert (green arrow). In contrast to Flp, the Ser integrase PhiC31 (bottom representations) leads to unidirectional integration, at least in the absence of an recombinase-directionality (RDF-)factor.<ref name = "karow">{{cite journal |doi=10.1517/14712598.2011.601293 |title=The therapeutic potential of phiC31 integrase as a gene therapy system |year=2011 |last1=Karow |first1=Marisa |last2=Calos |first2=Michele P |journal=Expert Opinion on Biological Therapy |volume=11 |issue=10 |pages=1287–96 |pmid=21736536|s2cid=2674915 }}</ref> Relative to Flp-RMCE, which requires two different ("heterospecific") ''FRT''-spacer mutants, the reaction partner (''att''B) of the first reacting ''att''P site is hit arbitrarily, such that there is no control over the direction the donor cassette enters the target (cf. the alternative products). Also different from [[Recombinase-mediated cassette exchange|Flp-RMCE]], several distinct RMCE targets cannot be mounted in parallel, owing to the lack of heterospecific (non-crossinteracting) ''att''P/''att''B combinations.
===Cre Recombinase===▼
[[Cre recombinase]] (Cre) is able to recombine specific sequences of DNA without the need for [[Enzyme#Cofactors|cofactors]]. The enzyme recognizes 34 [[base pair]] DNA sequences called [[Cre-Lox recombination#''loxP'' site|''loxP'']] ("locus of crossover in phage P1"). Depending on the orientation of target sites with respect to one another, Cre will integrate/excise or invert DNA sequences. Upon the excision (called "resolution" in case of a circular substrate) of a particular DNA region, normal gene expression is considerably compromised or terminated.<ref>{{cite journal |doi=10.1007/s10616-006-6550-0 |title=Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology |year=2006 |last1=Oumard |first1=André |last2=Qiao |first2=Junhua |last3=Jostock |first3=Thomas |last4=Li |first4=Jiandong |last5=Bode |first5=Juergen |journal=Cytotechnology |volume=50 |pages=93–108 |pmid=19003073 |issue=1–3 |pmc=3476001}}</ref>
Due to the pronounced resolution activity of Cre, one of its initial applications was the excision of ''lox''P-flanked ("floxed") genes leading to cell-specific gene knockout of such a floxed gene after Cre becomes expressed in the tissue of interest. Current technologies incorporate methods, which allow for both the spatial and temporal control of Cre activity. A common method facilitating the spatial control of genetic alteration involves the selection of a tissue-specific [[promotor (biology)|promoter]] to drive Cre expression. In order to control temporal activity of the excision reaction, forms of Cre which take advantage of various [[ligand]] binding domains have been developed. One successful strategy for inducing specific temporal Cre activity involves fusing the enzyme with a mutated ligand-binding ___domain for the human [[estrogen receptor]] (ERt). Upon the introduction of [[tamoxifen]] (an estrogen [[receptor antagonist]]), the Cre-ERt construct is able to penetrate the nucleus and induce targeted mutation. ERt binds tamoxifen with greater affinity than [[endogenous]] [[estrogens]], which allows Cre-ERt to remain [[cytoplasmic]] in animals untreated with tamoxifen. The temporal control of SSR activity by tamoxifen permits genetic changes to be induced later in [[embryogenesis]] and/or in adult tissues.<ref name = "leone" /> This allows researchers to bypass embryonic lethality while still investigating the function of targeted genes.
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[[Image:Fig3 Tag & Exchange.png|thumb|520px|'''A unified "tag-&-exchange strategy.''' Tag-and-exchange strategy relying on homologous recombination (HR; tagging step) followed by RMCE (SSR; exchange step). The figure illustrates analogous double-reciprocal crossover principles for HR and RMCE, the major difference being the dramatically different requirements for homologous sequences, which are in the kb-range for HR but as short as ~50 bp for SSRs]]
===Flp
In its natural host (S. cerevisiae) the [[FLP-FRT recombination|Flp/''FRT'']] system enables replication of a "2μ plasmid" by the inversion of a segment that is flanked by two identical, but oppositely oriented ''FRT'' sites ("flippase" activity). This inversion changes the relative orientation of replication forks within the plasmid enabling "rolling circle"
Based on the RMCE technology, a particular resource of pre-characterized ES-strains that lends itself to further elaboration has evolved in the framework of the EUCOMM (European Conditional Mouse Mutagenesis) program, based on the now established Cre- and/or Flp-based
The traditional, laborious "tag-and-exchange" procedures relied on two successive homologous recombination (HR-)steps, the first one ("HR1") to introduce a tag consisting of a selection marker gene. "HR2" was then used to replace the marker by the "GOI. In the first (
===PhiC31
Without much doubt, Ser
Contrary to the above Tyr recombinases, PhiC31-INT as such acts in a unidirectional manner, firmly locking in the donor vector at a genomically anchored target. An obvious advantage of this system is that it can rely on unmodified, native ''att''P (acceptor) and ''att''B donor sites. Additional benefits (together with certain complications) may arise from the fact that mouse and human genomes per se contain a limited number of endogenous targets (so called "''att''P-pseudosites"). Available information suggests that considerable DNA sequence requirements let the integrase recognize fewer sites than retroviral or even transposase-based integration systems
Exploiting the fact of specific (''att''P x ''att''B) recombination routes,
== Outlook and perspectives ==
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==External links==
*http://www.knockoutmouse.org/
*{{cite journal | last1 = Emes | first1 = RD | last2 = Goodstadt | first2 = L | last3 = Winter | first3 = EE | last4 = Ponting | first4 = CP | year = 2003 | title = Comparison of the genomes of human and mouse lays the foundation of genome zoology
[[Category:Genetic engineering]]
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