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.     

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.     

Non-contact positions impose site selectivity on Cre recombinase

A first step in Cre-mediated site-specific DNA recombination is binding to the two 13 bp repeats of the 34 bp site loxP. Several nucleotides within loxP do not directly contact the bound enzyme, yet mutation at two of these base pairs, at positions 11 and 12 in each repeat, results in a 100 000-fold reduction in recombination. To understand better how Cre selects DNA sequences for recombination, we combined DNA shuffling mutagenesis and a forward selection strategy to obtain Cre mutants that recombine at 100% efficiency a mutant loxK2 site carrying these dinucleotide changes. The role of the several mutations found in these Cre isolates was analyzed both in vivo and biochemically with purified enzymes. A single mutation at E262 accounts for most but not all of the enhanced activity at loxK2. Secondary mutations act in one or more of three ways: enhancement of loxK2 binding, accelerated synthesis of Cre in vivo or faster DNA recombination at the alternative spacer region present in loxK2. Systematic analysis of all 20 natural amino acids at position E262 shows that the naturally occurring glutamate residue at this position provides the optimal balance of efficiency of recombination at loxP and maximal discrimination against loxK2.     

Alteration of Cre recombinase site specificity by substrate-linked protein evolution.

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.     

Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites

The Cre-lox system is an important tool for genetic manipulation. To promote integrative reactions, two strategies using mutant lox sites have been developed. One is the left element/right element (LE/RE)-mutant strategy and the other is the cassette exchange strategy using heterospecific lox sites such as lox511 or lox2272. We compared the recombination efficiencies using these mutant lox sites in embryonic stem (ES) cells, and found that the combination of the LE/RE mutant and lox2272 showed high recombination efficiency and stability of the recombined structure. Taking advantage of this stability, we successfully integrated the cre gene into the mutant lox sites by Cre-mediated recombination. Germ line chimeric mice were produced from the cre-integrated ES cell clones, and Cre-expressing mouse lines were established. The inserted cre gene was stably maintained through the generations. This cre knock-in system using the LE/RE-lox2272 combination should be useful for the production of various cre mice for gene targeting or gene trapping.     

Genomic targeting with purified Cre recombinase.

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.     

Exchangeable gene trap using the Cre/mutated lox system.

The gene trap technique is a powerful approach for characterizing and mutating genes involved in mouse development. However, one shortcoming of gene trapping is the relative inability to induce subtle mutations. This problem can be overcome by introducing a knock-in system into the gene trap strategy. Here, we have constructed a new gene trap vector, pU-Hachi, employing the Cre-mutated lox system (Araki et al., 1997), in which a pair of mutant lox, lox71 and lox66, was used to promote targeted integrative reaction by Cre recombinase. The pU-Hachi carries splicing acceptor (SA)-lox71-internal ribosomal entry site (IRES)-beta-geo-pA-loxP-pA-pUC. By using this vector, we can carry out random insertional mutagenesis as the first step, and then we can replace the beta-geo gene with any gene of interest through Cre-mediated integration. We have isolated 109 trap clones electroporated with pU-Hachi, and analyzed their integration patterns by Southern blotting to select those carrying a single copy of the trap vector. By use of some of these clones, we have succeeded in exchanging the reporter gene at high efficiency, ranging between 20-80%. This integration system is also quite useful for plasmid rescue to recover flanking genomic sequences, because a plasmid vector sequence can be introduced even when the pUC sequence of the trap vector is lost through integration into the genome. Thus, this method, termed exchangeable gene trapping, has many advantages as the trapped clones can be utilized to express genes with any type of mutation.     

基于Cre重组酶体系的鸡卵清蛋白基因打靶载体的构建

利用胚胎干细胞基因打靶技术制备转基因鸡是研制鸡输卵管反应器的最佳技术路线。为建立基于Cre/loxp系统的鸡卵清蛋白基因(Ovalbumingene,OV)位点的双交换打靶载体系统,本研究克隆了鸡的OV基因7.8kb片段,并与克隆的内部核糖体进入位点(IRES)、人工合成的含有Cre重组酶识别位点变异体交换盒m2/loxp71EGFPloxp66,一起构建了含有Hsvtk负筛选标记的针对鸡卵清蛋白基因位点的敲入型共表达基因打靶载体pSSCm2/71EGFP66IRESOV7.8;以猪β干扰素基因(βInterferon,IFNβ)为目的基因构建了穿梭载体pMDm2/66MCSIFNMCSLoxp71,经过限制酶酶切及部分测序鉴定,所构建载体结构正确。进一步将它们共转化组成性表达Cre的细菌BM25.8,验证了loxp突变位点对重组反应的有效性     

Unmodified Cre recombinase crosses the membrane

Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous ‘protein transduction domain’ surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPα^fl/fl mice also led to excision of the ‘floxed’ C/EBPα gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

Generation of chickens expressing Cre recombinase

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.     

Alpha complementation in the Cre recombinase enzyme

The Cre-loxP system is increasingly exploited for spatial and temporal gene inactivation. Here we present a novel approach to achieve this goal of selective gene inactivation. Following the model of alpha complementation in the beta-galactosidase enzyme, where the enzyme is split into independent polypeptides which are able to associate and maintain the enzymatic activity, we divided the Cre recombinase into two independent polypeptides (one containing the NH(2) terminus (alpha) and a second one containing the COOH-terminus (beta)). Individually, the two polypeptides have no detectable activity. However, when coexpressed the polypeptides are able to associate, giving rise to Cre enzymatic activity, which optimally is as high as 30% of that seen with wildtype Cre recombinase in vitro. We present this strategy as a modification of the traditional Cre-loxP system, which could be used to obtain a highly specific recombination pattern by expressing the two halves under the control of separate promoters.     

Quasi-equivalence in site-specific recombinase structure and function: crystal structure and activity of trimeric Cre recombinase bound to a three-way Lox DNA junction

The crystal structure of a novel Cre-Lox synapse was solved using phases from multiple isomorphous replacement and anomalous scattering, and refined to 2.05 A resolution. In this complex, a symmetric protein trimer is bound to a Y-shaped three-way DNA junction, a marked departure from the pseudo-4-fold symmetrical tetramer associated with Cre-mediated LoxP recombination. The three-way DNA junction was accommodated by a simple kink without significant distortion of the adjoining DNA duplexes. Although the mean angle between DNA arms in the Y and X structures was similar, adjacent Cre trimer subunits rotated 29 degrees relative to those in the tetramers. This rotation was accommodated at the protein-protein and DNA-DNA interfaces by interactions that are "quasi-equivalent" to those in the tetramer, analogous to packing differences of chemically identical viral subunits at non-equivalent positions in icosahedral capsids. This structural quasi-equivalence extends to function as Cre can bind to, cleave and perform strand transfer with a three-way Lox substrate. The structure explains the dual recognition of three and four-way junctions by site-specific recombinases as being due to shared structural features between the differently branched substrates and plasticity of the protein-protein interfaces. To our knowledge, this is the first direct demonstration of quasi-equivalence in both the assembly and function of an oligomeric enzyme.     

Intein-mediated rapid purification of Cre recombinase

Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein. The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column. Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months.     

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.     

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.     

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.     

Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein

CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.