The widely accepted hypothesis postulates that the first spliceosomal introns originated from group II self-splicing introns. However, it is evident that not all spliceosomal introns in the nuclear genes of modern eukaryotes are inherited through vertical transfer of intronic sequences. Several phenomena contribute to the formation of new introns but their most common origin seems to be the insertion of transposable elements. Recent analyses have highlighted instances of mass gains of new introns from transposable elements. These events often coincide with an increase or change in the spliceosome\'s tolerance to splicing signals, including the acceptance of noncanonical borders. Widespread acquisitions of transposon-derived introns occur across diverse evolutionary lineages, indicating convergent processes. These events, though independent, likely require a similar set of conditions. These conditions include the presence of transposon elements with features enabling their removal at the RNA level as introns and/or the existence of a splicing mechanism capable of excising unusual sequences that would otherwise not be recognized as introns by standard splicing machinery. Herein we summarize those mechanisms across different eukaryotic lineages.

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.

BACKGROUND: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq).
RESULTS: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions.
CONCLUSIONS: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.

To illustrate the genomic and drug resistance traits of the Klebsiella pneumoniae Kpn_XM9, which harbors a transposon (Tn) As1 and was barely susceptible to ceftazidime-avibactam (CZA). Whole-genome sequencing, gene deletion, antimicrobial susceptibility, and conjugation tests were carried out to illustrate the traits of Kpn_XM9. As confirmed by whole-genome sequencing, the Kpn_XM9 harbored a 5,523,536 bp chromosome and five plasmids with lengths being 128,129, 196,512, 84,812, 43,695, and 5,596 bp, respectively. Plasmid p1_Kpn_XM9 (128,219 bp) contained four resistance genes, blaCTX-M-65, blaTEM-1B, rmtB, and two copies of blaKPC-2. Genes blaKPC-2 were bracketed by ISKpn17 and ISKpn16 within a new composite Tn3-like TnAs1. The two tandem repeats, positioned opposite each other, were spaced 93,447 bp apart in p1_Kpn_XM9. Kpn_XM9 belonged to K64 and sequence type (ST) 11. The Kpn_XM9 was resistant to amikacin, aztreonam, ticarcillin/clavulanic acid, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, tobramycin, ciprofloxacin, levofloxacin, doxycycline, minocycline, tigecycline, colistin, and trimethoprim/sulfamethoxazole; it was barely susceptible to CZA with a minimum inhibitory concentration of 8/4 µg/mL, which declined to 2/4 µg/mL after a 18,555 bp nucleotide was knocked out and one copy of blaKPC-2 was sustained on p1_Kpn_XM9. Kpn_XM9 had virulence genes encoding Types 1 and 3 fimbriae, four siderophores, and capsular polysaccharide anchoring protein but no genes upregulating capsular polysaccharide synthesis. The Kpn_XM9 presented a classical phenotype with extreme drug resistance. The emergence of double copies of blaKPC-2 in a single plasmid from the predominant ST11 K. pneumoniae represents a new therapeutic challenge.IMPORTANCEWith the wide use of ceftazidime-avibactam against carbapenem-resistant organisms, its resistance is increasingly documented; among the corresponding resistance mechanisms, mutations of blaKPC-2 or blaKPC-3 into other subtypes are dominant to date. However, more copies of blaKPC-2 may also greatly increase the minimum inhibitory concentration of ceftazidime-avibactam, which could be conferred by transposon As1 and insertion sequence 26 and should be of concern.

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.

Argonaute proteins are key constituents of small RNA-guided regulatory pathways. In crustaceans, members of the AGO subfamily of Argonaute proteins that play vital roles in immune defense are well studied, while proteins of the PIWI subfamily are less established. PmAgo4 of the black tiger shrimp, Penaeus monodon, though phylogenetically clustered with the AGO subfamily, has distinctive roles of the PIWI subfamily in safeguarding the genome from transposon invasion and controlling germ cell development. This study explored a molecular mechanism by which PmAgo4 regulates transposon expression in the shrimp germline. PmAgo4-associated small RNAs were co-immunoprecipitated from shrimp testis lysate using a PmAgo4-specific polyclonal antibody. RNA-seq revealed a majority of 26-27 nt long small RNAs in the PmAgo4-IP fraction suggesting that PmAgo4 is predominantly associated with piRNAs. Mapping of these piRNAs on nucleotide sequences of two gypsy and a mariner-like transposons of P. monodon suggested that most piRNAs were originated from the antisense strand of transposons. Suppression of PmAgo4 expression by a specific dsRNA elevated the expression levels of the three transposons while decreasing the levels of transposon-related piRNAs. Taken together, these results imply that PmAgo4 exerts its suppressive function on transposons by controlling the biogenesis of transposon-related piRNAs and thus, provides a defense mechanism against transposon invasion in shrimp germline cells.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

Transposons are integral genome constituents that can be domesticated for host functions, but they also represent a significant threat to genome stability. Transposon silencing is especially critical in the germline, which is dedicated to transmitting inherited genetic material. The small Piwi-interacting RNAs (piRNAs) have a deeply conserved function in transposon silencing in the germline. piRNA biogenesis and function are particularly well understood in Drosophila melanogaster, but some fundamental mechanisms remain elusive and there is growing evidence that the pathway is regulated in response to genotoxic and environmental stress. Here, we review transposon regulation by piRNAs and the piRNA pathway regulation in response to stress, focusing on the Drosophila female germline.

Novel active DNA transposons, such as Spy transposons from the PHIS superfamily, are identified through bioinformatics in this study. The native transposases cgSpy and cvSpy displayed transposition activities of approximately 85% and 35% compared to the hyperactive piggyBac transposase (hyPB). The cgSpy transposon showed unique characteristics, including a lack of overproduction inhibition and reduced efficiency for insertion sizes between 3.1 to 8.5 kb. Integration preferences of cgSpy are found in genes and regulatory regions, making it suitable for genetic manipulation. Evaluation in T-cell engineering demonstrated that cgSpy-mediated chimeric antigen receptor (CAR) modification is comparable to the PB system, indicating its potential utility in cell therapy. This study unveils the promising application of the active native transposase, Spy, from Colletes gigas, as a valuable tool for genetic engineering, particularly in T-cell manipulation.