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.     

Improved FLP Recombinase,FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–<Emphasis Type="Italic">lox</Emphasis> Mediated Site-Specific Gene Integration in Rice

Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.     

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.     

Functional characteristics of a highly specific integrase encoded by an LTR-retrotransposon

Background

The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5′-CGCGCg-3′ consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN.

Principal Findings

Here we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence.

Conclusion

This study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

Zinc finger recombinases with adaptable DNA sequence specificity

Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural 'pseudosites' are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.     

An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.     

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.     

Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems

DNA recombination reactions (site-specific and homologous) were monitored in the progeny of transgenic maize plants by bringing together two recombination substrates (docking sites and shuttle vectors) in the zygotes. In one combination of transgenic events, the recombination marker gene (yellow fluorescent protein gene, YFP) was activated in 1%-2% of the zygotes receiving both substrates. In other crosses, chimeric embryos and plants were identified, indicative of late recombination events taking place after the first mitotic division of the zygotes. The docking site structure remained unchanged; therefore, all recovered recombination events were classified as gene conversions. The recombinant YFP-r gene segregated as a single locus in subsequent generations. The recombination products showed evidence of homologous recombination at the 5' end of the YFP marker gene and recombinational rearrangements at the other end, consistent with the conclusion that DNA replication was involved in generation of the recombination products. Here, we demonstrate that maize zygotes are efficient at generating homologous recombination products and that the homologous recombination pathways may successfully compete with other possible DNA repair/recombination mechanisms such as site-specific recombination. These results indicate that maize zygotes provide a permissive environment for homologous recombination, offering a new strategy for gene targeting in maize.     

Altered directionality in the Cre-LoxP site-specific recombination pathway

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.     

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.     

Predicting knot and catenane type of products of site-specific recombination on twist knot substrates

Site-specific recombination on supercoiled circular DNA molecules can yield a variety of knots and catenanes. Twist knots are some of the most common conformations of these products, and they can act as substrates for further rounds of site-specific recombination. They are also one of the simplest families of knots and catenanes. Yet, our systematic understanding of their implication in DNA and important cellular processes such as site-specific recombination is very limited. Here, we present a topological model of site-specific recombination characterizing all possible products of this reaction on twist knot substrates, extending the previous work of Buck and Flapan. We illustrate how to use our model to examine previously uncharacterized experimental data. We also show how our model can help determine the sequence of products in multiple rounds of processive recombination and distinguish between products of processive and distributive recombinations.This model studies generic site-specific recombination on arbitrary twist knot substrates, a subject for which there is limited global understanding. We also provide a systematic method of applying our model to a variety of different recombination systems.     

Site-specific recombinases in genetic engineering: Modern in vivo technologies

A review was conducted over the current achievements in the area of site-specific recombinases (SSR) and their applications for manipulations with pro- and eukaryotic genomes. The principles of SSR functioning and the types of genetic rearrangements catalyzed by SSR were analyzed. The examples given in this review show the SSR potential to solve a wide range of basic and practical problems. To use different methods for solving these problems, however, would be more difficult or even impossible. The main directions for further developing the technology of site-specific recombination are the following: the use of SSR for a wider range of biological systems; generation of the SSR, which are characterized by strictly controlled expression in space and time; and the search for recombinases with new substrate specificity.     

DNA重组技术的研究综述

DNA重组技术,即DNA克隆技术的研究和运用是现代生物学发展的一个重要分支,是分子生物学发展的突出领域。本文介绍了DNA重组的类型及相关的生物学概念;综述了目前已报道的传统的酶切-连接经典克隆方法、位点特异性重组克隆方法、以及同源重组克隆方法,重点阐述了各自的原理、步骤、特点及实际应用等方面;最后归纳总结了各种方法的优缺点和应用范围,并对该技术的科研成果进行了回顾和对未来的研究进行了展望。     

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.     

An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.     

Targeted engineering of the Drosophila genome

The application of phiC31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004 1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phiC31. Three recent papers have further developed novel approaches that combine the phiC31-mediated DNA integration with the homologous recombination (HR)-based gene targeting 2 3 for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al. 4, the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group 6. All three use HR-based gene targeting to first implant a single or a pair of phiC31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phiC31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directedly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO, and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.     

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.     

Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.