Site-specific recombinase technology: Difference between revisions

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'''Site-specific recombinase technologies''' are [[genome engineering]] tools that depend on [[Recombinase|recombinase enzymes]] to replace targeted sections of DNA.

{{lead rewrite|date=December 2012}}

Nearly every human [[gene]] has a counterpart in the mouse (regardless of the fact that a minor set of [[Homology (biology)|orthologues]] had to follow species specific selection routes). This made the mouse the major model for elucidating the ways in which our genetic material encodes information. In the late 1980s gene targeting in murine [[embryonic stem cell|embryonic stem (ES-)cells]] enabled the transmission of mutations into the mouse germ line and emerged as a novel option to study the genetic basis of regulatory networks as they exist in the genome. Still, classical [[gene targeting]] proved to be limited in several ways as gene functions became irreversibly destroyed by the marker gene that had to be introduced for selecting recombinant ES cells. These early steps led to animals in which the mutation was present in all cells of the body from the beginning leading to complex phenotypes and/or early lethality. There was a clear need for methods to restrict these mutations to specific points in development and specific cell types. This dream became reality when groups in the USA were able to introduce bacteriophage and yeast-derived site-specific recombination (SSR-) systems into mammalian cells as well as into the mouse <ref>{{cite journal |first1=Brian |last1=Sauer |first2=Nancy |last2=Henderson |bibcode=1988PNAS...85.5166S |jstor=32380 |title=Site-Specific DNA Recombination in Mammalian Cells by the Cre Recombinase of Bacteriophage P1 |volume=85 |year=1988 |pages=5166–70 |journal=Proceedings of the National Academy of Sciences of the United States of America |doi=10.1073/pnas.85.14.5166 |pmid=2839833 |issue=14 |pmc=281709}}</ref><ref>{{cite journal |first1=Stephen |last1=O'Gorman |first2=Daniel T. |last2=Fox |first3=Geoffrey M. |last3=Wahl |bibcode=1991Sci...251.1351O |doi=10.1126/science.1900642 |jstor=2875533 |pmid=1900642 |title=Recombinase-mediated gene activation and site-specific integration in mammalian cells |year=1991 |journal=Science |volume=251 |issue=4999 |pages=1351–5}}</ref><ref name = "rajewski">{{cite journal |doi=10.1002/eji.200737819 |title=From a Dream to Reality |year=2007 |last1=Rajewsky |first1=Klaus |journal=European Journal of Immunology |volume=37 |pages=S134–7 |pmid=17972357}}</ref>

== History ==

==Site-specific recombinases: classification, properties and dedicated applications==

Nearly every human [[gene]] has a counterpart in the mouse (regardless of the fact that a minor set of [[Homology (biology)|orthologues]] had to follow species specific selection routes). This made the mouse the major model for elucidating the ways in which our genetic material encodes information. In the late 1980s gene targeting in murine [[embryonic stem cell|embryonic stem (ES-)cells]] enabled the transmission of mutations into the mouse germ line and emerged as a novel option to study the genetic basis of regulatory networks as they exist in the genome. Still, classical [[gene targeting]] proved to be limited in several ways as gene functions became irreversibly destroyed by the marker gene that had to be introduced for selecting recombinant ES cells. These early steps led to animals in which the mutation was present in all cells of the body from the beginning leading to complex phenotypes and/or early lethality. There was a clear need for methods to restrict these mutations to specific points in development and specific cell types. This dream became reality when groups in the USA were able to introduce bacteriophage and yeast-derived site-specific recombination (SSR-) systems into mammalian cells as well as into the mouse <ref>{{cite journal |first1=Brian |last1=Sauer |first2=Nancy |last2=Henderson |bibcode=1988PNAS...85.5166S |jstor=32380 |title=Site-Specific DNA Recombination in Mammalian Cells by the Cre Recombinase of Bacteriophage P1 |volume=85 |year=1988 |pages=5166–70 |journal=Proceedings of the National Academy of Sciences of the United States of America |doi=10.1073/pnas.85.14.5166 |pmid=2839833 |issue=14 |pmc=281709}}</ref><ref>{{cite journal |first1=Stephen |last1=O'Gorman |first2=Daniel T. |last2=Fox |first3=Geoffrey M. |last3=Wahl |bibcode=1991Sci...251.1351O |doi=10.1126/science.1900642 |jstor=2875533 |pmid=1900642 |title=Recombinase-mediated gene activation and site-specific integration in mammalian cells |year=1991 |journal=Science |volume=251 |issue=4999 |pages=1351–5}}</ref><ref name = "rajewski">{{cite journal |doi=10.1002/eji.200737819 |title=From a Dream to Reality |year=2007 |last1=Rajewsky |first1=Klaus |journal=European Journal of Immunology |volume=37 |pages=S134–7 |pmid=17972357}}</ref>

==Site-specific recombinases: classificationClassification, 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. |journal=Developmental Cell |volume=6 |pages=7–28 |pmid=14723844 |issue=1}}</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 |first3=K. |last4=Rubin |first4=G. M. |journal=Proceedings of the National Academy of Sciences |volume=108 |issue=34 |pages=14198–203 |pmid=21831835}}</ref>

[[File:Classification_of_site-specific_recombinases_according_to_mechanism.png|thumb|755x755px|'''''Figure 1: 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 |doilast2=10Bode|first2=J.1096/fj.11-186940 |year=2011|title=Site-specific recombinases: From tag-and-target- to tag-and-exchange-based genomic modifications |year=2011 |last1=Turan |first1=S. |last2=Bode |first2=J. |journal=The FASEB Journal |volume=25 |issue=12 |pages=4088–107 |doi=10.1096/fj.11-186940|pmid=21891781|last1=Turan|first1=S.}}</ref>]]

The founding member of the YR family is the [[lambda integrase]], encoded by [[Bacteriophage| 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 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 enzymesof listedthesse in Fig. 1enzymes recombine two target sites, which are either identical (subfamily A1) or distinct (phage-derived enzymes in A2, B1 and B2).<ref name="turan" /> Whereas for A1 these sites have individual designations ("''FRT''" in case of Flp-recombinase, "''lox''P" for Cre-recombinase), the terms "''att''P" and "''att''B" (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).<ref>{{cite journal |doi=10.1515/BC.2000.103 |title=The Transgeneticists Toolbox: Novel Methods for the Targeted Modification of Eukaryotic Genomes |year=2000 |last1=Bode |first1=Jürgen |last2=Schlake |first2=Thomas |last3=Iber |first3=Michaela |last4=Schübeler |first4=Dirk |last5=Seibler |first5=Jost |last6=Snezhkov |first6=Evgeney |last7=Nikolaev |first7=Lev |journal=Biological Chemistry |volume=381 |issue=9–10 |pmid=11076013 |pages=801–13}}</ref> Note that for Flp there is an alternative, 48 bp site available with three arms, each accommodating a Flp unit (a so called "protomer"). ''att''P- and ''att''B-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:

[[Image:Classification of site-specific recombinases according to mechanism.png|''Figure 1: Classification of site-specific recombinases'']]

'''''Figure 1: 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 |doi=10.1096/fj.11-186940 |title=Site-specific recombinases: From tag-and-target- to tag-and-exchange-based genomic modifications |year=2011 |last1=Turan |first1=S. |last2=Bode |first2=J. |journal=The FASEB Journal |volume=25 |issue=12 |pages=4088–107 |pmid=21891781}}</ref>

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).<ref name="turan"/> Whereas for A1 these sites have individual designations ("''FRT''" in case of Flp-recombinase, "''lox''P" for Cre-recombinase), the terms "''att''P" and "''att''B" (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).<ref>{{cite journal |doi=10.1515/BC.2000.103 |title=The Transgeneticists Toolbox: Novel Methods for the Targeted Modification of Eukaryotic Genomes |year=2000 |last1=Bode |first1=Jürgen |last2=Schlake |first2=Thomas |last3=Iber |first3=Michaela |last4=Schübeler |first4=Dirk |last5=Seibler |first5=Jost |last6=Snezhkov |first6=Evgeney |last7=Nikolaev |first7=Lev |journal=Biological Chemistry |volume=381 |issue=9–10 |pmid=11076013 |pages=801–13}}</ref> Note that for Flp there is an alternative, 48 bp site available with three arms, each accommodating a Flp unit (a so called "protomer"). ''att''P- and ''att''B-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 ''att''P x ''att''B recombination crossovers can only occur between these complementary partners in processes that lead to two different products (''att''P x ''att''B → ''att''R + ''att''L) 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).

[[File:Fig2A.png|thumb|right|600px|'''''Figure 2:''' '''Recombination patterns depending on recombinase (sub-)family and target-site orientation.'''

GOI, "gene of interest"; [+/-], a positive-negative selection marker such as the hygtk-fusion gene. Note that interaction of two identical substrate sites (''lox''PloxP x ''lox''PloxP or ''FRT'' x ''FRT'') leads to products of the same composition, whereas recombination of two non-identical educts leads to two different hybrid sites (''att''PattP x ''att''BattB → ''att''RattR + ''att''LattL)'']]

== Reaction routes enabled by reversibly acting Tyr-Recombinases and a unidirectional Ser-Integrase ==

Fig. 2 shows, in sections A-B, theThe 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> Of note is the way reversible Flp-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}}</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 ("causes recombination") is able to recombine specific sequences of DNA without the need for cofactors. The enzyme recognizes 34 base pair DNA sequences called ''lox''P (“locus of crossover in phage P1”). Depending on the orientation of target sites with respect to one another, Cre will integrate/excise (Fig. 2, Section A) or invert DNA sequences (Fig.2, Section B). 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 (Fig.2, section "A") 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. If placed under the control of such a promoter, in turn allows for the localized expression of Cre in certain tissues. As an example, Leone et al. have placed the transcription unit under the control of the regulatory sequences of the [[myelin]] proteolipid protein (PLP) gene, leading to induced removal of targeted gene sequences in [[oligodendrocytes]] and [[Schwann cells]].<ref name = "leone">{{cite journal |doi=10.1016/S1044-7431(03)00029-0 |title=Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells |year=2003 |last1=Leone |first1=Dino P |last2=Genoud |first2=S.Téphane |last3=Atanasoski |first3=Suzana |last4=Grausenburger |first4=Reinhard |last5=Berger |first5=Philipp |last6=Metzger |first6=Daniel |last7=MacKlin |first7=Wendy B |last8=Chambon |first8=Pierre |last9=Suter |first9=Ueli |journal=Molecular and Cellular Neuroscience |volume=22 |issue=4 |pages=430–40 |pmid=12727441 }}</ref> The specific DNA fragment recognized by Cre remains intact in cells, which do not express the PLP gene; this in turn facilitates empirical observation of the localized effects of genome alterations in the myelin sheath that surround nerve fibers in the [[central nervous system]] (CNS) and the [[peripheral nervous system]] (PNS).<ref name=koenning>{{cite journal |doi=10.1523/JNEUROSCI.1069-12.2012 |title=Myelin Gene Regulatory Factor is Required for Maintenance of Myelin and Mature Oligodendrocyte Identity in the Adult CNS |year=2012 |last1=Koenning |first1=M. |last2=Jackson |first2=S. |last3=Hay |first3=C. M. |last4=Faux |first4=C. |last5=Kilpatrick |first5=T. J. |last6=Willingham |first6=M. |last7=Emery |first7=B. |journal=Journal of Neuroscience |volume=32 |issue=36 |pages=12528–42 |pmid=22956843}}</ref> Selective Cre expression has been achieved in many other cell types and tissues as well.

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.

Recent extensions of these general concepts led to generating the "Cre-zoo", i.e. collections of hundreds of mouse strains for which defined genes can be deleted by targeted Cre expression.<ref name="rajewski"/>

[[Image:Fig3 Tag & Exchange.png|thumb|right|520px|'''''Figure 3:''' '''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 Recombinase===

Line 51 ⟶ 49:

===PhiC31 Integrase===

Without much doubt, Ser integrases are the current tools of choice for integrating transgenes into a restricted number of well-understood genomic acceptor sites that mostly (but not always) mimic the phage ''att''P site in that they attract an ''att''B-containing donor vector. At this time the most prominent member is PhiC31-INT (Fig. 2, sections A-C in the lower panel) with proven potential in the context of human and mouse genomes.

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 openig its career as a superior carrier vehicle for the transport and insertion at a number of well established genomic sites, some of which with so called "safe-harbor" properties.<ref name="karow" />

Exploiting the fact of specific (''att''P x ''att''B) recombination routes, [[Recombinase-mediated cassette exchange|RMCE]] becomes possible without requirements for synthetic, heterospecific ''att''-sites. This obvious advantage, however comes at the expense of certain shortcomings, such as lack of control about the kind or directionality of the entering (donor-) cassette (indicated in Fig. 2, section C).<ref name="turan" /> Further restrictions are imposed by the fact that irreversibility does not permit standard [[Recombinase-mediated cassette exchange|multiplexing-RMCE]] setups including "serial RMCE" reactions, i.e., repeated cassette exchanges at a given genomic ''locus''.

== Outlook and perspectives ==