Site-specific recombinase technology: Difference between revisions

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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]]s (ESCs) 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 ESCs. 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|doi-access=free }}</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|doi-access=free }}</ref>

==Classification, properties and dedicated applications==

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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]]&nbsp;/&nbsp;[[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 these enzymes 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, [[Cre-Lox recombination#''loxP'' site|''loxP'']] 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|s2cid=36479502 }}</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:

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===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. Placement of Cre under control of such a promoter results in localized, tissue-specific expression. 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 |s2cid=624620 }}</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|pmc=3752083 }}</ref> Selective Cre expression has been achieved in many other cell types and tissues as well.