DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ϕw39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.     

Site-specific recombination strategies for engineering actinomycete genomes

The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific loxLE and loxRE sites was similar to its activity on wild-type loxP sites. Moreover, an apramycin resistance marker flanked by the loxLERE sites was eliminated from the Streptomyces coelicolor M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from Streptomyces sp. strain Tü6071, Streptomyces cinnamonensis A519, and Streptomyces aureofaciens Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.     

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.     

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.     

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.     

PhiC31 integrase facilitates genetic approaches combining multiple recombinases

Homologous and site-specific DNA recombination has revolutionized genetic engineering. The reliability of recombinases such as Cre and FLP has allowed scientists to design complex strategies to study gene function in mammals. However, the retention of recombination sites in the genome limits the use of Cre and FLP recombinases in subsequent modifications. Access to additional recombinases in the ES cell toolbox would enormously widen the number of possibilities to manipulate the genome. In the method presented here, we combine the use of PhiC31, a site-specific integrase, with FLP to obtain site-specific insertion and replacement in pre-inserted docking sites in the genome of mouse ES cells. This method allows for the integration of any sequence of interest in a pre-defined locus, leaving Cre recombinase available for downstream applications. The selection strategy is based on a silent selection marker activated by a plasmid-delivered promoter, making the integration system highly reliable and reducing the need for extensive molecular screens. This article describes how to create "dockable" mouse embryonic stem (ES) cell lines, integrate incoming vectors, and analyze the resulting clones. Current applications of this technology are also discussed.     

Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: Implications for applied site-specific recombination

The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional gene expression. To test whether these recombinases are active in Plasmodium falciparum we constructed gene knockouts that contain sequences recognised as templates for site-specific recombination. We tested the ability of FLP and Cre recombinases, expressed conditionally in P. falciparum, to mediate deletion of the human dihydrofolate reductase (hdhfr) drug resistance gene. We show that Cre recombinase is capable of efficient removal of hdhfr by site-specific recombination. In contrast, FLP recombinase is very inefficient, even at the optimum temperature of 30 °C for this enzyme. These results demonstrate that Cre recombinase can be utilised in P. falciparum for deletion of specific sequences such as drug resistance genes. This can be exploited for recycling of drug resistance cassettes and for the design of specific recombination events in P. falciparum.     

Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Cre重组酶结构与功能的研究进展

Cre/loxP定位重组系统来源于噬菌体P₁,由Cre重组酶和loxP位点两部分组成。在Cre重组酶的介导下,设定的DNA片段可以被切除,可以发生倒位,亦可造成定点的整合。由于其作用方式高效简单,Cre/loxP定位重组系统已在特定基因的删除、基因功能的鉴定、外源基因的整合、基因捕获及染色体工程等方面得到了有效的利用,在转基因的酵母、植物、昆虫、哺乳动物的体内外DNA重组方面成为一个有力的工具。这里就Cre重组酶的结构、功能及该定位重组系统的应用等方面的研究进行了综述。     

Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems

Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).     

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.     

A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants

SUMMARY: The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-microm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system.     

Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems,and their system specificity in mice

DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.     

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.     

An engineered lox sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision.

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.     

A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.     

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.     

A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

With current plant transformation methods (Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted ‘position effects’ caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.     

Excision of selectable genes from transgenic goat cells by a protein transducible TAT-Cre recombinase

The Cre/loxP site-specific recombination system is a widely used tool for genetic engineering of mammalian genomes. Recombination of loxP-modified alleles is often induced by introduction of foreign DNA vector expressing Cre into the cells. But the introduced DNA vector has the potential to integrate into the genome of the cells and continuous expression of Cre recombinase from the foreign vector has the potential to yield cytotoxicity and genotoxicity in various cells. In this study, we investigate the possibility of overcoming this limitation by using a cell-permeable TAT-Cre recombinase. We found that TAT-Cre treatment of transgenic goat fibroblast cells did not compromise the development competency of reconstructed embryos by using these TAT-Cre-treated cells as nuclear donor in nuclear transfer. Finally, we obtained two live cloned goats where a selectable gene cassette was removed. Our work not only provided an efficient protein transduction-based system for removing selectable genes from transgenic goats, but also presented strong evidence that no severe damage was made to the host cells during the process of protein transduction.