Transformation-associated Recombination Cloning Research Articles

Comparative analysis and classification of highly divergent mouse rDNA units based on their intergenic spacer (IGS) variability.

Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5'ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Rapid Construction of Recombinant PDCoV Expressing an Enhanced Green Fluorescent Protein for the Antiviral Screening Assay Based on Transformation-Associated Recombination Cloning in Yeast.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that mainly causes diarrhea and death in suckling piglets and also has the potential for cross-species transmission, threatening public health. However, there is still no effective vaccine or drug to prevent PDCoV infection. In order to accelerate the development of antiviral drugs, we established a high-throughput screening platform using a novel genome editing technology called transformation-associated recombination cloning in yeast. The recombinant PDCoV and PDCoV reporter virus expressing enhanced green fluorescent protein were both rapidly rescued with stable genealogical characteristics during passage. Further study demonstrated that the reporter virus can be used for high-throughput screening of antiviral drugs with a Z-factor of 0.821-0.826. Then, a medicine food homology compound library was applied, and we found that three compounds were potential antiviral reagents. In summary, we have established a fast and efficient reverse genetic system of PDCoV, providing a powerful platform for the research of antiviral drugs.

Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine.

Transformation-associated recombination (TAR) cloning represents a unique tool to selectively and efficiently recover a given chromosomal segment up to several hundred kb in length from complex genomes (such as animals and plants) and simple genomes (such as bacteria and viruses). The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. In this review, we summarize multiple applications of the pioneering TAR cloning technique, developed previously for complex genomes, for functional, evolutionary, and structural studies, and extended the modified TAR versions to isolate biosynthetic gene clusters (BGCs) from microbes, which are the major source of pharmacological agents and industrial compounds, and to engineer synthetic viruses with novel properties to design a new generation of vaccines. TAR cloning was adapted as a reliable method for the assembly of synthetic microbe genomes for fundamental research. In this review, we also discuss how the TAR cloning in combination with HAC (human artificial chromosome)- and CRISPR-based technologies may contribute to the future.

Functional expression of foreign magnetosome genes in the alphaproteobacterium Magnetospirillum gryphiswaldense.

Magnetosomes of magnetotactic bacteria (MTB) consist of structurally perfect, nano-sized magnetic crystals enclosed within vesicles of a proteo-lipid membrane. In species of Magnetospirillum, biosynthesis of their cubo-octahedral-shaped magnetosomes was recently demonstrated to be a complex process, governed by about 30 specific genes that are comprised within compact magnetosome gene clusters (MGCs). Similar, yet distinct gene clusters were also identified in diverse MTB that biomineralize magnetosome crystals with different, genetically encoded morphologies. However, since most representatives of these groups are inaccessible by genetic and biochemical approaches, their analysis will require the functional expression of magnetosome genes in foreign hosts. Here, we studied whether conserved essential magnetosome genes from closely and remotely related MTB can be functionally expressed by rescue of their respective mutants in the tractable model Magnetospirillum gryphiswaldense of the Alphaproteobacteria. Upon chromosomal integration, single orthologues from other magnetotactic Alphaproteobacteria restored magnetosome biosynthesis to different degrees, while orthologues from distantly related Magnetococcia and Deltaproteobacteria were found to be expressed but failed to re-induce magnetosome biosynthesis, possibly due to poor interaction with their cognate partners within multiprotein magnetosome organelle of the host. Indeed, co-expression of the known interactors MamB and MamM from the alphaproteobacterium Magnetovibrio blakemorei increased functional complementation. Furthermore, a compact and portable version of the entire MGCs of M. magneticum was assembled by transformation-associated recombination cloning, and it restored the ability to biomineralize magnetite both in deletion mutants of the native donor and M. gryphiswaldense, while co-expression of gene clusters from both M. gryphiswaldense and M. magneticum resulted in overproduction of magnetosomes. IMPORTANCE We provide proof of principle that Magnetospirillum gryphiswaldense is a suitable surrogate host for the functional expression of foreign magnetosome genes and extended the transformation-associated recombination cloning platform for the assembly of entire large magnetosome gene cluster, which could then be transplanted to different magnetotactic bacteria. The reconstruction, transfer, and analysis of gene sets or entire magnetosome clusters will be also promising for engineering the biomineralization of magnetite crystals with different morphologies that would be valuable for biotechnical applications.

Construction of lipopeptide mono-producing Bacillus strains and comparison of their antimicrobial activity

Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides, including iturin, surfactin, and plipastatin, and it is challenging to discriminate each cyclic lipopeptide's antimicrobial activity directly. In this study, a chassis strain Bacillus subtilis 1A751 WR without cyclic lipopeptides operons was constructed by CRISPR/Cas9. The iturin gene cluster of B. amyloliquefaciens HYM-12 was captured and assembled with sfp and degQ genes using transformation-associated recombination (TAR). It was then integrated into the chassis strain to obtain an iturin mono-producing strain that could synthesize six iturin isoforms. The frameshift sfp gene of strain 1A751 Δpps was repaired by a scarless gene knock-in method to generate a surfactin mono-producing strain. Antimicrobial activity experiments indicated that the extracts of iturin, surfactin, and our previously constructed plipastatin mono-producing strains exhibited powerful inhibitory effects toward pathogenic bacteria. Interestingly, iturin showed significant antifungal activity, but surfactin and plipastatin exhibited weak antifungal activity. Compared with amphotericin B, iturin has a solid and durable antifungal activity. This study demonstrates that TAR cloning is efficient for cloning large gene clusters, proves the differences in the antimicrobial activity of three cyclic lipopeptides, and lays a theoretical basis for further elucidating their antimicrobial mechanism.

Development of a rapid reverse genetics system for feline coronavirus based on TAR cloning in yeast.

Reverse genetics has become an indispensable tool to gain insight into the pathogenesis of viruses and the development of vaccines. The yeast-based synthetic genomics platform has demonstrated the novel capabilities to genetically reconstruct different viruses. In this study, a transformation-associated recombination (TAR) system in yeast was used to rapidly rescue different strains of feline infectious peritonitis virus, which causes a deadly disease of cats for which there is no effective vaccine. Using this system, the viruses could be rescued rapidly and stably without multiple cloning steps. Considering its speed and ease of manipulation in virus genome assembly, the reverse genetics system developed in this study will facilitate the research of the feline coronaviruses pathogenetic mechanism and the vaccine development.

Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating pandemic worldwide. Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic, and scientists all over the world have made great efforts to this end. However, manipulation of the SARS-CoV-2 should be performed in the biosafety level 3 laboratory. This makes experiments complicated and time-consuming. Therefore, a safer system for working with this virus is urgently needed. Here, we report the construction of plasmid-based, non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae. Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes. Using SARS-CoV-2 replicons, the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed, and the half-maximal effective concentration (EC50) value of remdesivir and E64-D was estimated by different quantification methods respectively, indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12250-021-00369-9.

The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning

The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.

Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.

In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline.

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.

Clone of plipastatin biosynthetic gene cluster by transformation-associated recombination technique and high efficient expression in model organism Bacillus subtilis

Plipastatin, a cyclic lipopeptide, exhibits inhibitory activity against filamentous fungi and plays an important role in the prevention of plant diseases, and post-harvest preservation of fruits and vegetables. However, the application of plipastatin has been hampered by low yields in natural strains, while chemical synthesis is not feasible because of its complex chemical structure. In this study, a scarless genetic modification method was applied to construct a heterologous expression host (Bacillus subtilis 1A751 Δpps) by knocking out the natural plipastatin genes from B. subtilis strain 1A751. The core genes for plipastatin biosynthesis from B. amyloliquefaciens HYM12 were captured and assembled with sfp and degQ using transformation-associated recombination (TAR) cloning. The resultant gene cluster was introduced into B. subtilis 1A751 Δpps to generate strain B. subtilis 1A751 Δpps amyE::ppsA–E + sfp + degQ. Its fermentation products were analyzed and identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The results showed that the gene cluster for plipastatin synthesis was expressed successfully. This is the first time three gene fragments with significantly different DNA sizes (38.4, 0.3 and 0.8 kb) have been assembled by the TAR technique, thus we simultaneously established a method to express recombined large fragments in B. subtilis.

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ∼0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

Analysis of the 9p21.3 sequence associated with coronary artery disease reveals a tendency for duplication in a CAD patient.

Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed ‘duplication blocks’. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ∼50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer.

A Genomics-Based Approach Identifies a Thioviridamide-Like Compound with Selective Anticancer Activity

Thioviridamide is a structurally novel ribosomally synthesized and post-translational modified peptide (RiPP) produced by Streptomyces olivoviridis NA005001. It is characterized by a structure that features a series of thioamide groups and possesses potent antiproliferative activity in cancer cell lines. Its unusual structure allied to its promise as an anticancer compound led us to investigate the diversity of thioviridamide-like pathways across sequenced bacterial genomes. We have isolated and characterized three diverse members of this family of natural products. This characterization is supported by transformation-associated recombination cloning and heterologous expression of one of these compounds, thiostreptamide S4. Our work provides an insight into the diversity of this rare class of compound and indicates that the unusual N-terminus of thioviridamide is not introduced biosynthetically but is instead introduced during acetone extraction. A detailed analysis of the biological activity of one of the newly discovered compounds, thioalbamide, indicates that it is highly cytotoxic to cancer cells, while exhibiting significantly less activity toward a noncancerous epithelial cell line.

Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.

PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete.

The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.

Cloning, Stability, and Modification of Mycoplasma hominis Genome in Yeast.

Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent cloning and subsequent engineering of other mycoplasma genomes in yeast opens new possibilities for studies of the genomes of genetically intractable organisms. Here, we report the successful one-step cloning of the M.hominis PG21 genome in yeast using the transformation-associated recombination (TAR) cloning method. At low passages, the M.hominis genome cloned into yeast displayed a conserved size. However, after ∼60 generations in selective media, this stability was affected, and large degradation events were detected, raising questions regarding the stability of large heterologous DNA molecules cloned in yeast and the need to minimize host propagation. Taking these results into account, we selected early passage yeast clones and successfully modified the M.hominis PG21 genome using the CRISPR/Cas9 editing tool, available in Saccharomyces cerevisiae. Complete M.hominis PG21 genomes lacking the adhesion-related vaa gene were efficiently obtained.