Converting an FRT-Tagged Gene into a Fluorescent Protein Gene Fusion by Flp-Mediated Site-Specific Recombination.

This protocol uses conditional plasmids carrying the open reading frame (orf) of either superfolder green fluorescent protein (sfGFP) or monomeric Cherry (mCherry) fused to a flippase (Flp) recognition target (FRT) site. In cells expressing the Flp enzyme, site-specific recombination between the plasmid-borne FRT and an FRT "scar" in a target gene in the bacterial chromosome results in chromosomal integration of the plasmid with the concomitant in-frame fusion of the target gene to the fluorescent protein orf. This event can be positively selected using an antibiotic-resistance marker (kan or cat) present on the plasmid. This method is slightly more laborious than generating the fusion directly by recombineering and has the limitation that the selectable marker is no longer removable. However, it has the advantage that it can be more readily integrated in mutational studies, allowing conversion of in-frame deletions resulting from Flp-mediated excision of a drug-resistance cassette (e.g., all those of the "Keio collection") into fluorescent protein fusions. Furthermore, in studies that require that the amino-terminal moiety of the hybrid protein keeps its biological activity, presence of the FRT "linker" sequence at the fusion junction makes it less likely for the fluorescent domain to sterically interfere with the folding of the amino-terminal domain.

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in Caenorhabditis elegans.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

Exploration of DNA processing features unravels novel properties of ICE conjugation in Gram-positive bacteria.

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOBT. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOBT relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOBT relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOBT relaxase.

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution.

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Generation of markerless and multiple-gene knockout in Glaesserella parasuis based on natural transformation and Flp recombinase.

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (KanR) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the KanR cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the KanR cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of ΔhsdR, ΔneuA2, ΔespP2, Δapd, and ΔnanH were constructed. In addition, a five-gene knockout mutant of ΔhsdRΔneuA2ΔespP2ΔapdΔnanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. KEY POINTS: • Flp recombinase excised the KanR gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout forG. parasuis. • The technique will promote the understanding of Glässer's disease pathogens.

Dual recombinase action in the normal and neoplastic mammary gland epithelium.

We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.

A DNA Inversion System in Eukaryotes Established via Laboratory Evolution.

DNA inversion is a type of site-specific recombination system that plays an important role in the generation of genetic diversity and phenotypic adaptation by programmed rearrangements in bacteria. However, no such inversion system exhibiting a strong directionality bias has been identified or developed in eukaryotes yet. Here, using directed evolution of Rci recombinase, a tyrosine recombinase from a bacterial DNA inversion system, we identified a mutant Rci8 with a ratio of inversion/deletion up to ∼4320 in yeast. Based on Rci8 recombinase and sfxa101 sites, we have established a DNA inversion system in yeast and mammalian cells, enabling specificity for DNA inversions between inverted sites over deletions between directly repeated sites. Our results validated that the reversible DNA inversion system can act as an on/off transcriptional switch. Moreover, we demonstrate that the inversion system can also work on linear chromosomes. The eukaryotic DNA inversion system would provide a new tool for fields of genetic circuits, cellular barcoding, and synthetic genomes.

Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria.

Transcriptional rewiring is the regulation of different target genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

Gene Assembly in Agrobacterium via Nucleic Acid Transfer Using Recombinase Technology (GAANTRY).

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

FLP-Mediated Site-Specific Gene Integration in Rice.

Enabling precise gene integration is important for installing traits in the plants. One of the practical methods of achieving precise gene integration is by using the yeast FLP-FRT recombination system that is efficient in directing DNA integration into the "engineered" genomic sites. The critical parameters of this method include the use of the thermostable version of FLP protein and the promoter trap design to select site-specific integration clones. The resulting transgenic plants display stable expression that is transmitted to the progeny. Therefore, FLP-mediated site-specific integration method could be used for trait engineering in the crop plants or testing gene functions in the model plants.

HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

A Lambda Red and FLP/FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose.

Komagataeibacter xylinus has received increasing attention as an important microorganism for the conversion of several carbon sources to bacterial cellulose (BC). However, BC productivity has been impeded by the lack of efficient genetic engineering techniques. In this study, a lambda Red and FLP/FRT-mediated site-specific recombination system was successfully established in Komagataeibacter xylinus. Using this system, the membrane bound gene gcd, a gene that encodes glucose dehydrogenase, was knocked out to reduce the modification of glucose to gluconic acid. The engineered strain could not produce any gluconic acid and presented a decreased bacterial cellulose (BC) production due to its restricted glucose utilization. To address this problem, the gene of glucose facilitator protein (glf; ZMO0366) was introduced into the knockout strain coupled with the overexpression of the endogenous glucokinase gene (glk). The BC yield of the resultant strain increased by 63.63-173.68%, thus reducing the production cost.

Cell-Type-Specific Gene Inactivation and In Situ Restoration via Recombinase-Based Flipping of Targeted Genomic Region.

Conditional gene inactivation and restoration are powerful tools for studying gene functions in the nervous system and for modeling neuropsychiatric diseases. The combination of the two is necessary to interrogate specific cell types within defined developmental stages. However, very few methods and animal models have been developed for such purpose. Here we present a versatile method for conditional gene inactivation and in situ restoration through reversibly inverting a critical part of its endogenous genomic sequence by Cre- and Flp-mediated recombinations. Using this method, we generated a mouse model to manipulate Mecp2, an X-linked dosage-sensitive gene whose mutations cause Rett syndrome. Combined with multiple Cre- and Flp-expressing drivers and viral tools, we achieved efficient and reliable Mecp2 inactivation and restoration in the germline and several neuronal cell types, and demonstrated phenotypic reversal and prevention on cellular and behavioral levels in male mice. This study not only provides valuable tools and critical insights for Mecp2 and Rett syndrome, but also offers a generally applicable strategy to decipher other neurologic disorders.SIGNIFICANCE STATEMENT Studying neurodevelopment and modeling neurologic disorders rely on genetic tools, such as conditional gene regulation. We developed a new method to combine conditional gene inactivation and restoration on a single allele without disturbing endogenous expression pattern or dosage. We applied it to manipulate Mecp2, a gene residing on X chromosome whose malfunction leads to neurologic disease, including Rett syndrome. Our results demonstrated the efficiency, specificity, and versatility of this new method, provided valuable tools and critical insights for Mecp2 function and Rett syndrome research, and offered a generally applicable strategy to investigate other genes and genetic disorders.

Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases.

The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-specific recombinases (SSRs) for genome editing applications. Previous work has revealed key residues for target site selectivity in the Cre/loxP and the related Dre/rox recombinase systems. However, enzymes in which these residues were changed to the respective counterpart only showed weak activity on the foreign target site. Here, we use molecular modeling and dynamics simulation techniques to comprehensively explore the mechanisms by which these residues determine target recognition in the context of their flanking regions in the protein-DNA interface, and we establish a structure-based rationale for the design of improved recombination activities. Our theoretical models reveal that nearest-neighbors to the specificity-determining residues are important players for enhancing SSR activity on the foreign target site. Based on the established rationale, we design new Cre variants with improved rox recombination activities, which we validate experimentally. Our work provides new insights into the target recognition mechanisms of Cre-like recombinases and represents an important step towards the rational design of SSRs for applied genome engineering.

DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages.

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.

Efficient Transgenesis in Caenorhabditis elegans Using Flp Recombinase-Mediated Cassette Exchange.

The application of CRISPR technology has greatly facilitated the creation of transgenic Caenorhabditis elegans lines. However, methods to insert multi-kilobase DNA constructs remain laborious even with these advances. Here, I describe a new approach for introducing large DNA constructs into the C. elegans genome at specific sites using a combination of Flp and Cre recombinases. The system utilizes specialized integrated landing sites that express GFP ubiquitously flanked by single loxP, FRT, and FRT3 sites. DNA sequences of interest are inserted into an integration vector that contains a sqt-1 self-excising cassette and FRT and FRT3 sites. Plasmid DNA is injected into the germline of landing site animals. Transgenic animals are identified as Rol progeny, and the sqt-1 marker is subsequently excised with heat shock Cre expression. Integration events were obtained at a rate of approximately one integration per three injected F0 animals-a rate substantially higher than any current approach. To demonstrate the robustness of the approach, I compared the efficiency of the Gal4/UAS, QF (and QF2)/QUAS, tetR(and rtetR)/tetO, and LexA/lexO bipartite expression systems by assessing expression levels in combinations of driver and reporter GFP constructs and a direct promoter GFP fusion each integrated at multiple sites in the genome. My data demonstrate that all four bipartite systems are functional in C. elegans Although the new integration system has several limitations, it greatly reduces the effort required to create single-copy insertions at defined sites in the C. elegans genome.

Site-Specific Recombination with Inverted Target Sites: A Cautionary Tale of Dicentric and Acentric Chromosomes.

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

PRISM: A Progenitor-Restricted Intersectional Fate Mapping Approach Redefines Forebrain Lineages.

Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages-Shh and Cux2.

Generation of an Fsp1 (fibroblast-specific protein 1)-Flpo transgenic mouse strain.

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1-Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast-specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1-Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.

Genetic lineage tracing with multiple DNA recombinases: A user's guide for conducting more precise cell fate mapping studies.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase-mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate-related biological phenomena in the life sciences.