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.     

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.     

Site-specific recombinases as tools for heterologous gene integration

Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.     

Site-specific gene targeting for gene expression in eukaryotes

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.     

Genome engineering using site-specific recombinases

The targeted modification of the mammalian genome has a variety of applications in research, medicine, and biotechnology. Site-specific recombinases have become significant tools in all of these areas. Conditional gene targeting using site-specific recombinases has enabled the functional analysis of genes, which cannot be inactivated in the germline. The site-specific integration of adeno-associated virus, a major gene therapy vehicle, relies on the recombinase activity of the viral rep proteins. Site-specific recombinases also allow the precise integration of open reading frames encoding pharmaceutically relevant proteins into highly active gene loci in cell lines and transgenic animals. These goals have been accomplished by using a variety of genetic strategies but only a few recombinase proteins. However, the vast repertoire of recombinases, which has recently become available as a result of large-scale sequencing projects, may provide a rich source for the development of novel strategies to precisely alter mammalian genomes.     

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.     

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.     

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.     

Identification and characterization of the dif Site from Bacillus subtilis

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6 degrees counterclockwise from the B. subtilis terminus of replication (172 degrees ), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPbeta-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains.     

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.     

Selection on the genes of Euplotes crassus Tec1 and Tec2 transposons: evolutionary appearance of a programmed frameshift in a Tec2 gene encoding a tyrosine family site-specific recombinase

The Tec1 and Tec2 transposons of the ciliate Euplotes crassus carry a gene for a tyrosine-type site-specific recombinase. The expression of the Tec2 gene apparently uses a programmed +1 frameshift. To test this hypothesis, we first examined whether this gene has evolved under purifying selection in Tec1 and Tec2. Each element carries three genes, and each has evolved under purifying selection for the function of its encoded protein, as evidenced by a dearth of nonsynonymous changes. This distortion of divergence is apparent in codons both 5' and 3' of the frameshift site. Thus, Tec2 transposons have diverged from each other while using a programmed +1 frameshift to produce recombinase, the function of which is under purifying selection. What might this function be? Tyrosine-type site-specific recombinases are extremely rare in eukaryotes, and Tec elements are the first known eukaryotic type II transposons to encode a site-specific recombinase. Tec elements also encode a widespread transposase. The Tec recombinase might function in transposition, resolve products of transposition (bacterial replicative transposons use recombinase or resolvase to separate joined replicons), or provide a function that benefits the ciliate host. Transposons in ciliated protozoa are removed from the macronucleus, and it has been proposed that the transposons provide this "excisase" activity.     

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.     

Mammalian genomes contain active recombinase recognition sites

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.     

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.     

Recent innovations in tissue-specific gene modifications in the mouse

Annotating the functions of individual genes in in vivo contexts has become the primary task of mouse genetics in the post-genome era. In addition to conventional approaches using transgenic technologies and gene targeting, the recent development of conditional gene modification techniques has opened novel opportunities for elucidating gene function at the level of the whole mouse to individual tissues or cell types. Tissue-specific gene modifications in the mouse have been made possible using site-specific DNA recombinases and conditional alleles. Recent innovations in this basic technology have facilitated new types of experiments, revealing novel insights into mammalian embryology. In this review, we focus on these recent innovations and new technical issues that impact the success of these conditional gene modification approaches.     

Evolution of programmable zinc finger-recombinases with activity in human cells

Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (Rec(ZF)s) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in approximately 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)s may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.     

Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

Summary: A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.     

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.     

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.