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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 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:▼
▲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]] /
Our table extends the selection of the conventional SSR systems and groups these according to their performance. All of thesse 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, "''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:▼
▲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>
===Cre
[[Cre recombinase]] (
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
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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