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Transposable elements (TEs) are indispensable for human development, with critical functions in pluripotency and embryogenesis. TE sequences also contribute to human pathologies, especially cancer, with documented activities as cis / trans transcriptional regulators, as sources of non-coding RNAs, and as mutagens that disrupt tumor suppressors. Despite this knowledge, little is known regarding the involvement of TE-derived genes (TEGs) in tumor pathogenesis. Here, systematic analyses of TEG expression across human cancer reveal a prominent role for pogo TE derived with KRAB domain (POGK). We show that POGK acts as a tumor suppressor in triple-negative breast cancer (TNBC) cells and that it couples with the co-repressor TRIM28 to directly block the transcription of ribosomal genes RPS16 and RPS29, in turn causing widespread inhibition of ribosomal biogenesis. We report that POGK undergoes deactivation by isoform switching in clinical TNBC, altogether revealing its exapted activities in tumor growth control. [Display omitted] • Widespread, substantial deregulation of TE-derived genes in human cancer • POGK exerts tumor-suppressive functions in TNBC • POGK suppresses global ribosomal biogenesis by inhibiting RPS16 and RPS29 • Inactivation of POGK growth-suppressive functions in TNBC by exon skipping Transposable element sequences occupy >50% of the human genome and serve evolutionarily critical roles in development. Here, Tu et al. show that the transposable-element-derived gene POGK partners with TRIM28 to inhibit transcription of ribosomal genes RPS16 and RPS29, thereby disrupting ribosomal biogenesis and aiding in triple-negative breast cancer growth control. [ABSTRACT FROM AUTHOR]

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements. [ABSTRACT FROM AUTHOR]

The piggyBac transposon/transposase system has been explored for long‐term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI‐L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB‐GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA‐MB‐231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI‐L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac‐based stable expression of therapeutic genes. [ABSTRACT FROM AUTHOR]

ABSTRACTTransposases are mobile genetic elements that move within and between genomes, promoting genomic plasticity in microorganisms. In marine microbial communities, the abundance of transposases increases with depth, but the reasons behind this trend remain unclear. Our analysis of metagenomes from the Tara Oceans and Malaspina Expeditions suggests that a particle-associated lifestyle is the main covariate for the high occurrence of transposases in the deep ocean, and this trend holds true for individual genomes as well as in a community-wide sense. We observed a strong and depth-independent correlation between transposase abundance and the presence of biofilm-associated genes, as well as the prevalence of secretory enzymes. This suggests that mobile genetic elements readily propagate among microbial communities within crowded biofilms. Furthermore, we show that particle association positively correlates with larger genome size, which is in turn associated with higher transposase abundance. Cassette sequences associated with transposons are enriched with genes related to defense mechanisms, which are more highly expressed in the deep sea. Thus, while transposons spread at the expense of their microbial hosts, they also introduce novel genes and potentially benefit the hosts in helping to compete for limited resources. Overall, our results suggest a new understanding of deep ocean particles as highways for gene sharing among defensively oriented microbial genomes.IMPORTANCEGenes can move within and between microbial genomes via mobile genetic elements, which include transposases and transposons. In the oceans, there is a puzzling increase in transposase abundance in microbial genomes as depth increases. To gain insight into this trend, we conducted an extensive analysis of marine microbial metagenomes and metatranscriptomes. We found a significant correlation between transposase abundance and a particle-associated lifestyle among marine microbes at both the metagenome and genome-resolved levels. We also observed a link between transposase abundance and genes related to defense mechanisms. These results suggest that as microbes become densely packed into crowded particles, mobile genes are more likely to spread and carry genetic material that provides a competitive advantage in crowded habitats. This may enable deep sea microbes to effectively compete in such environments.

Background: Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT's recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. Results: Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. Conclusion: The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads. [ABSTRACT FROM AUTHOR]

Incidence of Streptococcus dysgalactiae subspecies equisimilis (SDSE) bacteremia is increasing in the Kyoto-Shiga region of Japan. We retrospectively analyzed clinical features of SDSE bacteremia and conducted comparative genomic analyses of isolates collected from 146 bacteremia episodes among 133 patients during 2005–2021. Of those patients, 7.7% required vasopressor support, and 7.0% died while in the hospital. The prevalence of isolates resistant to erythromycin, minocycline, and clindamycin increased from 8.6% during 2005–2017 to 21.6% during 2018–2021. Our genomic analysis demonstrated that sequence type 525 and clonal complex 25 were predominant in SDSE isolates collected during 2018–2021. In addition, those isolates had acquired 2 antimicrobial-resistance genes, ermB and tetM, via Tn916-like integrative and conjugative elements (ICEs). Phylogenetic analysis revealed clonal distribution of Tn916-like ICEs in SDSE isolates. Our findings suggest that Tn916-like ICEs contributed to the emergence and recent increase of multidrug-resistant SDSE bacteremia in this region of Japan.

The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ∼3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1–3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods. • Improved productivity and stability of product quality during cell line development using transposase. • Interrogation of integration sites and their effect on the genome in recombinant bioproduction cell lines. • Chromosome painting and transgene locus amplification (TLA) for genetic characterization of bioproduction cell lines. • Mechanisms and strategies to prevent transgenic silencing during cell line development. [ABSTRACT FROM AUTHOR]

Nearly all eukaryotes carry DNA transposons of the Robertson's Mutator (Mu) superfamily, a widespread source of genome instability and genetic variation. Despite their pervasive impact on host genomes, much remains unknown about the evolution of these transposons. Transposase recognition of terminal inverted repeats (TIRs) is thought to drive and constrain coevolution of MuDR transposase genes and TIRs. To address the extent of this relationship and its impact, we compared separate phylogenies of TIRs and MuDR gene sequences from Mu elements in the maize genome. Five major clades were identified. As expected, most Mu elements were bound by highly similar TIRs from the same clade (homomorphic type). However, a subset of elements contained dissimilar TIRs derived from divergent clades. These "heteromorphs" typically occurred in multiple copies indicating active transposition in the genome. In addition, analysis of internal sequences showed that exchanges between elements having divergent TIRs produced new mudra and mudrb gene combinations. In several instances, TIR homomorphs had been regenerated within a heteromorph clade with retention of distinctive internal MuDR sequence combinations. Results reveal that recombination between divergent clades facilitates independent evolution of transposase (mudra), transposase-binding targets (TIRs), and capacity for insertion (mudrb) of active Mu elements. This mechanism would be enhanced by the preference of Mu insertions for recombination-rich regions near the 5' ends of genes. We suggest that cycles of recombination give rise to alternating homo-and heteromorph forms that enhance the diversity on which selection for Mu fitness can operate. [ABSTRACT FROM AUTHOR]

HUH endonucleases of the Rep (replication protein) class mediate the replication of highly diverse plasmids and viral genomes across all domains of life. HUH transposases have independently evolved from Reps, giving rise to three major transposable element groups: the prokaryotic insertion sequences IS200/IS605 and IS91/ISCR, and the eukaryotic Helitrons. Here, I present Replitrons, a second group of eukaryotic transposons encoding Rep HUH endonuclease. Replitron transposases feature a Rep domain with one catalytic Tyr (Y1) and an adjacent domain that may function in oligomerization, contrasting with Helitron transposases that feature Rep with two Tyr (Y2) and a fused helicase domain (i.e., RepHel). Protein clustering found no link between Replitron transposases and described HUH transposases, and instead recovered a weak association with Reps of circular Rep-encoding single-stranded (CRESS) DNA viruses and their related plasmids (pCRESS). The predicted tertiary structure of the transposase of Replitron-1, the founding member of the group that is active in the green alga Chlamydomonas reinhardtii, closely resembles that of CRESS-DNA viruses and other HUH endonucleases. Replitrons are present in at least three eukaryotic supergroups and reach high copy numbers in nonseed plant genomes. Replitron DNA sequences feature short direct repeats at, or potentially near, their termini. Finally, I characterize copy-and-paste de novo insertions of Replitron-1 using long-read sequencing of C. reinhardtii experimental lines. These results support an ancient and evolutionarily independent origin of Replitrons, in line with other major groups of eukaryotic transposons. This work expands the known diversity of both transposons and HUH endonucleases in eukaryotes. [ABSTRACT FROM AUTHOR]

Mobile genetic elements (MGEs) are the DNA sequences capable of moving inside the host genome. As a result of ongoing mutation processes and low natural selection pressure, the evolutionary diversity of MGEs is constantly increasing. Eukaryotic DNA transposons represent a very large and diverse group of MGEs. This work was focused on the study of the pogo DNA transposons in three genomes of jellyfish in the genus Aurelia: Aurelia aurita, Aurelia coerulea, and Aurelia aurita complex sp. In this work, the local alignment method (BLASTn) was used to search for the pogo group MGEs. Multiple amino-acid sequence alignment was performed using MAFFT. It was demonstrated that the majority of pogo elements were represented by the Fot/Fot-like family elements. Two new subfamilies, auFLE1 and auFLE2, were identified in this group. The Jelly group including two elements was also detected in jellyfish. No potentially functional DNA transposons were found among the detected pogo elements. Analysis of the identified pogo elements showed that no active DNA transposons were preserved in the studied jellyfish genomes: all elements are damaged by mutations or have deletions and are not represented by full-size copies. This allowed us to suggest that the activity peak for these transposons took place relatively long ago. [ABSTRACT FROM AUTHOR]

Background: Identification of transposons without close homologs is still a difficult task. IS630/Tc1/mariner transposons, classified into a superfamily, are probably the most widespread DNA transposons in nature. Tc1/mariner transposons have been discovered in animals, plants, and filamentous fungi, however, not in yeast. Results: In the present study, we report the discovery of two intact Tc1 transposons in yeast and filamentous fungi, respectively. The first one, named Tc1-OP1 (DD40E), represents Tc1 transposons in Ogataea parapolymorpha. The second one, named Tc1-MP1 (DD34E), represents Tc1 transposons in the Rhizopodaceae and Mucoraceae families. As a homolog of Tc1-OP1 and Tc1-MP1, IS630-AB1 (DD34E) was discovered as an IS630 transposon in Acinetobacter spp. Conclusion: Tc1-OP1 is not only the first reported Tc1 transposon in yeast, but also the first reported nonclassical Tc1 transposon. Tc1-OP1 is the largest of IS630/Tc1/ mariner transposons reported to date and significantly different from others. Notably, Tc1-OP1 encodes a serine-rich domain and a transposase, extending the current knowledge of Tc1 transposons. The phylogenetic relationships of Tc1-OP1, Tc1- MP1 and IS630-AB1 indicated that these transposons had evolved from a common ancestor. Tc1-OP1, Tc1-MP1 and IS630-AB1 can be used as reference sequences to facilitate the identification of IS630/Tc1/mariner transposons. More Tc1/mariner transposons will be identified in yeast, following our discovery. [ABSTRACT FROM AUTHOR]

Transposable elements (DNA transposons and retrotransposons) are DNA sequences that have the ability to change their position within a genome. They are suggested to play one of the key roles in adaptive and evolutionary processes. One of the most studied groups of DNA transposons is the ITm infraclass and, in particular, the Tc1/mariner superfamily. In the present study, the abundance, structure, and evolution of the Tc1/mariner DNA transposons in the jellyfish Aurelia aurita was examined. It was demonstrated that in jellyfish, the predominant proportion of Tc1/mariner elements was represented by the TLE family. A new subfamily of TLE elements (termed Aurum) was identified. In addition, in the Visitor family, two groups of elements, VS-aura and VS-beplau, were identified, which were probably separate subfamilies. Analysis of the structure and diversity of Tc1/mariner elements shows that Tc1/mariner transposons in the jellyfish genome are currently at the stage of degradation and elimination. Almost all elements are deleted or have structural changes and, accordingly, do not have potentially functional copies. [ABSTRACT FROM AUTHOR]

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined. [ABSTRACT FROM AUTHOR]

Mariner-like elements (MLEs) are promising tools for gene cloning, gene expression, and gene tagging. We have characterized two MLE transposons from moso bamboo, Ppmar1 and Ppmar2. Ppmar2, is smaller in size and has higher natural activities, thus making it a more potential genomic tool compared to Ppmar1. Using a two-component system consisting of a transposase expression cassette and a non-autonomous transposon cotransformed in yeast, we investigated the transposition activity of Ppmar2 and created hyperactive transposases. Five out of 19 amino acid mutations in Ppmar2 outperformed the wild-type in terms of catalytic activities, especially with the S347R mutant having 6.7-fold higher transposition activity. Moreover, 36 yeast mutants with single-gene deletion were chosen to screen the effects of the host factors on Ppmar2NA transposition. Compared to the control strain (his3Δ), the mobility of Ppmar2 was greatly increased in 9 mutants and dramatically decreased in 7 mutants. The transposition ability in the efm1Δ mutant was 15-fold higher than in the control, while it was lowered to 1/66 in the rtt10Δ mutant. Transcriptomic analysis exhibited that EFM1 defection led to the significantly impaired DDR2, HSP70 expression and dramatically boosted JEN1 expression, whereas RTT10 defection resulted in significantly suppressed expression of UTP20, RPA190 and RRP5. Protein methylation, chromatin and RNA transcription may affect the Ppmar2NA transposition efficiency in yeast. Overall, the findings provided evidence for transposition regulation and offered an alternative genomic tool for moso bamboo and other plants. [ABSTRACT FROM AUTHOR]

Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g. schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1 , also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure. [ABSTRACT FROM AUTHOR]

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months. • Chinese hamster ovary cells. • Cell line development. • Transposase. • Acceleration. • Monoclonal antibody. [ABSTRACT FROM AUTHOR]

In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.

Mutants of Micrococcus luteus strain ATCC49732 lacking the yellow pigment sarcinaxanthin were observed at an unexpectedly high frequency and the molecular basis was investigated. PCR probing revealed complete deletion of the crt biosynthetic operon in 11/14 mutants. Inverse PCR was used to identify a common breakpoint 35 kb downstream from crt precisely at the end of the right inverted repeat (IRR) of a partial ISMlu8 element that lies between two inversely oriented full-length ISMlu2. A total of three different breakpoints 5′ to crt were found with the sequence CTAG one bp 5′ to each novel junction. Analysis of 35 genomic sites with single ISMlu8 insertions showed that ISMlu8 transposase has high specificity for CTAG, implicating its key role in formation of the Δ crt deletions. No downstream deletion endpoints were observed at an immediately adjacent ISMlu8 with a nearly identical IRR in the same orientation and slightly closer to the crt operon, indicating that access of ISMlu8 transposase to the ISMlu2-flanked ISMlu8 IRR is greatly enhanced by the surrounding oppositely oriented ISMlu2s. The association of high frequency genomic rearrangement with this distinctive natural configuration of ISs from two different IS families offers a new insight into IS element evolutionary potential. [ABSTRACT FROM AUTHOR]

Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources., (© 2024 American Institute of Chemical Engineers.)

Transposases are mobile genetic elements that move within and between genomes, promoting genomic plasticity in microorganisms. In marine microbial communities, the abundance of transposases increases with depth, but the reasons behind this trend remain unclear. Our analysis of metagenomes from the Tara Oceans and Malaspina Expeditions suggests that a particle-associated lifestyle is the main covariate for the high occurrence of transposases in the deep ocean, and this trend holds true for individual genomes as well as in a community-wide sense. We observed a strong and depth-independent correlation between transposase abundance and the presence of biofilm-associated genes, as well as the prevalence of secretory enzymes. This suggests that mobile genetic elements readily propagate among microbial communities within crowded biofilms. Furthermore, we show that particle association positively correlates with larger genome size, which is in turn associated with higher transposase abundance. Cassette sequences associated with transposons are enriched with genes related to defense mechanisms, which are more highly expressed in the deep sea. Thus, while transposons spread at the expense of their microbial hosts, they also introduce novel genes and potentially benefit the hosts in helping to compete for limited resources. Overall, our results suggest a new understanding of deep ocean particles as highways for gene sharing among defensively oriented microbial genomes.IMPORTANCEGenes can move within and between microbial genomes via mobile genetic elements, which include transposases and transposons. In the oceans, there is a puzzling increase in transposase abundance in microbial genomes as depth increases. To gain insight into this trend, we conducted an extensive analysis of marine microbial metagenomes and metatranscriptomes. We found a significant correlation between transposase abundance and a particle-associated lifestyle among marine microbes at both the metagenome and genome-resolved levels. We also observed a link between transposase abundance and genes related to defense mechanisms. These results suggest that as microbes become densely packed into crowded particles, mobile genes are more likely to spread and carry genetic material that provides a competitive advantage in crowded habitats. This may enable deep sea microbes to effectively compete in such environments., Competing Interests: The authors declare no conflict of interest.

Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

During the last decade a wide range of single-cell and single-nucleus next-generation sequencing techniques have been developed, which revolutionized detection of rare cell populations, enabling creation of comprehensive cell atlases of complex organs and tissues. State-of-the-art methods do not only allow classical transcriptomics of individual cells but also comprise a number of epigenetic approaches, including assessment of chromatin accessibility by single-nucleus Assay for Transposase Accessible Chromatin ATAC-seq (snATAC-seq). The snATAC-seq assay detects "open chromatin," a term for low nucleosome occupancy of genomic regions, which is a prerequisite for effective transcription factor binding. Information about open chromatin at the single-nucleus level helps to recognize epigenetic changes, sometimes before transcription of respective genes occurs. snATAC-seq detects cellular heterogeneity in otherwise still transcriptionally and/or morphologically homogeneous cell populations. Chromatin accessibility assays may be used to detect epigenetic changes in cardiac lineages during heart development, chromatin landscape changes during aging, and epigenetic alterations in heart diseases. Here, we provide an optimized protocol for snATAC-seq of murine hearts. We describe isolation of single nuclei from snap-frozen hearts, provide hints for preparation of libraries suitable for snATAC-seq next-generation sequencing (NGS) using the Chromium 10× platform, and give general recommendations for downstream analysis using conventional bioinformatic pipelines and packages. The protocol should serve as a beginner's guide to generate high-quality snATAC-seq datasets and to perform chromatin accessibility analysis of individual heart-derived cell nuclei., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

In this chapter, the methodologies are outlined for generating CAR-T from PBMCs using transposon engineering. Additionally, some methods and guidance related to basic functional and phenotypic analysis are described. This methodology can be applied to manufacture and assess chimeric antigen receptors for preclinical applications targeting a variety of molecules., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRISPR/Cas-based gene-editing technologies have emerged as one of the most transformative tools in genome science over the past decade, providing unprecedented possibilities for both fundamental and translational research. Following the initial wave of innovations for gene knock-out, epigenetic/RNA modulation, and nickase-mediated base-editing, recent efforts have pivoted towards long-sequence gene editing- specifically, the insertion of large fragments (>1 kb) into the endogenous genome. In this review, we survey the development of these CRISPR/Cas-based sequence insertion methodologies in conjunction with the emergence of novel families of editing enzymes, such as transposases, single-stranded DNA-annealing proteins, recombinases, and integrases. Despite facing a number of challenges, this field continues to evolve rapidly and holds the potential to catalyze a new wave of revolutionary biomedical applications.

The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associates with AAA+ ATPase TnsC and catalyzes donor DNA cleavage and integration remains ambiguous. In this study, we demonstrate that TniQ-dCas9 fusion can direct site-specific transposition by TnsB/TnsC in ShCAST. TnsB is a 3′-5′ exonuclease that specifically cleaves donor DNA at the end of the terminal repeats and integrates the left end prior to the right end. The nucleotide preference and the cleavage site of TnsB are markedly different from those of the well-documented MuA. We also find that TnsB/TnsC association is enhanced in a half-integration state. Overall, our results provide valuable insights into the mechanism and application expansion of CRISPR-mediated site-specific transposition by TnsB/TnsC. [Display omitted] • A TniQ-dCas9 fusion is able to guide site-specific transposition by TnsB/TnsC • TnsB is a site-specific 3′-5′ exonuclease that could cleave donor DNA at the terminus • Recognition by TnsB is essential for cleavage and efficient transposition • TnsC filaments are a prerequisite for its association with TnsB Zeng et al. show that the natural component Cas12k can be substituted with TniQ-dCas9 fusion, retaining the ability to catalyze site-specific transposition. They investigate the biochemical properties of TnsB and its association with TnsC, which provides important insights into the mechanism of transposition mediated by the V-K CAST. [ABSTRACT FROM AUTHOR]

• The complete genome of Woronichinia naegeliana WA131 was determined from a microcystin-containing CyanoHAB in a US Pacific NW freshwater lake (2018). • The larger genome size of 7.9 Mpb, compared to other bloom-forming cyanobacteria, is mostly attributable to insertion elements and their transposase genes, which are often present in high copy number. Pseudogenes are also relatively abundant, but most of these are transposases. • The genome contains no genes for cyanotoxins or the common taste-and-odor compounds. Gene clusters for the production of anabaenopeptins, cyanopeptolins, microginins and some post-translationally modified peptides are present. • The genome of this planktonic, non-diazotrophic cyanobacterium contains genes commonly found in other bloom-forming cyanobacteria, but surprisingly lacks genes for nitrite and nitrate reductases, making this cyanobacterium dependent on reduced and organic forms of nitrogen. • In the US Pacific NW, Woronichinia naegeliana was a common component of freshwater HABs across the period 2007–2019, often being the dominant or subdominant cyanobacterium. Monitoring in the U.S. state of Washington across the period 2007–2019 showed that Woronichinia has been present in many lakes state-wide. This cyanobacterium was commonly dominant or sub-dominant in cyanobacterial blooms in the wet temperate region west of the Cascade Mountains. In these lakes, Woronichinia often co-existed with Microcystis, Dolichospermum and Aphanizomenon flos-aquae and the cyanotoxin microcystin has often been present in those blooms, although it has not been known whether Woronichinia is a toxin producer. We report the first complete genome of Woronichinia naegeliana WA131, assembled from the metagenome of a sample collected from Wiser Lake, Washington, in 2018. The genome contains no genes for cyanotoxin biosynthesis or taste-and-odor compounds, but there are biosynthetic gene clusters for other bioactive peptides, including anabaenopeptins, cyanopeptolins, microginins and ribosomally produced, post-translationally modified peptides. Genes for photosynthesis, nutrient acquisition, vitamin synthesis and buoyancy that are typical of bloom-forming cyanobacteria are present, although nitrate and nitrite reductase genes are conspicuously absent. However, the 7.9 Mbp genome is 3–4 Mbp larger than those of the above-mentioned frequently co-existing cyanobacteria. The increased genome size is largely due to an extraordinary number of insertion sequence elements (transposons), which account for 30.3% of the genome and many of which are present in multiple copies. The genome contains a relatively large number of pseudogenes, 97% of which are transposase genes. W. naegeliana WA131 thus seems to be able to limit the potentially deleterious effects of high rates of recombination and transposition to the mobilome fraction of its genome. [Display omitted] [ABSTRACT FROM AUTHOR]

Incidence of Streptococcus dysgalactiae subspecies equisimilis (SDSE) bacteremia is increasing in the Kyoto-Shiga region of Japan. We retrospectively analyzed clinical features of SDSE bacteremia and conducted comparative genomic analyses of isolates collected from 146 bacteremia episodes among 133 patients during 2005-2021. Of those patients, 7.7% required vasopressor support, and 7.0% died while in the hospital. The prevalence of isolates resistant to erythromycin, minocycline, and clindamycin increased from 8.6% during 2005-2017 to 21.6% during 2018-2021. Our genomic analysis demonstrated that sequence type 525 and clonal complex 25 were predominant in SDSE isolates collected during 2018-2021. In addition, those isolates had acquired 2 antimicrobial-resistance genes, ermB and tetM, via Tn916-like integrative and conjugative elements (ICEs). Phylogenetic analysis revealed clonal distribution of Tn916-like ICEs in SDSE isolates. Our findings suggest that Tn916-like ICEs contributed to the emergence and recent increase of multidrug-resistant SDSE bacteremia in this region of Japan.

Background: Identification of transposons without close homologs is still a difficult task. IS630/Tc1/mariner transposons, classified into a superfamily, are probably the most widespread DNA transposons in nature. Tc1/mariner transposons have been discovered in animals, plants, and filamentous fungi, however, not in yeast., Results: In the present study, we report the discovery of two intact Tc1 transposons in yeast and filamentous fungi, respectively. The first one, named Tc1-OP1 (DD40E), represents Tc1 transposons in Ogataea parapolymorpha . The second one, named Tc1-MP1 (DD34E), represents Tc1 transposons in the Rhizopodaceae and Mucoraceae families. As a homolog of Tc1-OP1 and Tc1-MP1, IS630-AB1 (DD34E) was discovered as an IS630 transposon in Acinetobacter spp., Conclusion: Tc1-OP1 is not only the first reported Tc1 transposon in yeast, but also the first reported nonclassical Tc1 transposon. Tc1-OP1 is the largest of IS630/Tc1/mariner transposons reported to date and significantly different from others. Notably, Tc1-OP1 encodes a serine-rich domain and a transposase, extending the current knowledge of Tc1 transposons. The phylogenetic relationships of Tc1-OP1, Tc1-MP1 and IS630-AB1 indicated that these transposons had evolved from a common ancestor. Tc1-OP1, Tc1-MP1 and IS630-AB1 can be used as reference sequences to facilitate the identification of IS630/Tc1/mariner transposons. More Tc1/mariner transposons will be identified in yeast, following our discovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chang, Duan, Li, Yau, Liu, Cui, Xue, Bu, Hu and Gao.)