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ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5'-GXXT-3' sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The transposons of the hAT superfamily are the most widespread transposons ever known. SLEEPER genes encode domesticated transposases from the hAT superfamily, which may have lost their transposable functions during long-term evolution and transformed into host proteins that regulate plant growth and development., Results: This study identified 162 members of the SLEEPER gene family from Brassica napus. These members are widely distributed on 19 chromosomes, mainly in the Cn subgenome, and have promoters with various cis-acting elements related to hormone regulation, abiotic stress, and growth and development regulation. Most of the genes in this family contain similar conserved domains and motifs, and the closer the genes are distributed on evolutionary branches, the more similar their structures are. Transcriptome sequencing performed on tissues at different growth stages from B. napus line 3529 indicated that these genes had different expression patterns, and nearly half of the genes were not detectably expressed in all samples., Conclusions: This study investigated the gene structure, expression patterns, evolutionary features, and gene localization of the SLEEPER family members to confirm the significance of these genes in the growth of B. napus, providing a reference for the study of transposon domestication and outstanding genetic resources for the genetic improvement of B. napus., (© 2024. The Author(s).)

Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes., (© 2024. The Author(s).)

Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is being increasingly used to study gene regulation. However, major analytical gaps limit its utility in studying gene regulatory programs in complex diseases. In response, MOCHA (Model-based single cell Open CHromatin Analysis) presents major advances over existing analysis tools, including: 1) improving identification of sample-specific open chromatin, 2) statistical modeling of technical drop-out with zero-inflated methods, 3) mitigation of false positives in single cell analysis, 4) identification of alternative transcription-starting-site regulation, and 5) modules for inferring temporal gene regulatory networks from longitudinal data. These advances, in addition to open chromatin analyses, provide a robust framework after quality control and cell labeling to study gene regulatory programs in human disease. We benchmark MOCHA with four state-of-the-art tools to demonstrate its advances. We also construct cross-sectional and longitudinal gene regulatory networks, identifying potential mechanisms of COVID-19 response. MOCHA provides researchers with a robust analytical tool for functional genomic inference from scATAC-seq data., (© 2024. The Author(s).)

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.

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.

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., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored., Methods: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC)., Results: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spf ash mouse following elimination of residual endogenous murine OTC., Conclusions: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset., (© 2024 John Wiley & Sons, Ltd.)

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies., Competing Interests: Declaration of interests E.U. is an Advisory Board member for Phialogics and has sponsored research projects with Gilead and BMS. Z.I. is an inventor on patents related to Sleeping Beauty and MC technology. M.H. is listed as inventor on patent applications and granted patents related to CAR technologies and transposon-based gene transfer that are, in part licensed to industry. M.H. is a co-founder and equity owner of T-CURX GmbH, Würzburg, Germany. M.H. and E.U. are inventors on patents related to CAR and MC technology. T.B., P.W., W.S.W., and E.U. are inventors on patents related to optimized CAR designs. T.B., P.W., and E.U. are inventors on non-viral gene-editing technologies of NK cells., (Copyright © 2024. Published by Elsevier Inc.)

Foodborne diseases are major public health problems globally. Metagenomics has emerged as a widely used tool for pathogen screening. In this study, we conducted an updated Tn5 transposase-assisted RNA/DNA hybrid co-tagmentation (TRACE) library construction approach. To address the detection of prevalent known foodborne viruses and the discovery of unknown pathogens, we employed both specific primers and oligo-T primers during reverse transcription. The method was validated using clinical samples confirmed by RT-qPCR and compared with standard RNA-seq library construction methods. The mapping-based approach enabled the retrieval of nearly complete genomes (>95%) for the majority of virus genome segments (86 out of 88, 97.73%), with a mean coverage depth of 21,494.53× (ranging from 77.94× to 55,688.58×). Co-infection phenomena involving prevalent genotypes of Norovirus with Astrovirus and Human betaherpesvirus 6B were observed in two samples. The updated TRACE-seq exhibited superior performance in viral reads percentages compared to standard RNA-seq library preparation methods. This updated method has expanded its target pathogens beyond solely Norovirus to include other prevalent foodborne viruses. The feasibility and potential effectiveness of this approach were then evaluated as an alternative method for surveilling foodborne viruses, thus paving the way for further exploration into whole-genome sequencing of viruses.

Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination 1-4 . These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6 ). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans 7 . Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility 8 , phage susceptibility 9 , and host immunity 10 . Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

In recent years, molecular biology-based diagnostic techniques have made remarkable strides and are now extensively utilized in clinical practice, providing invaluable insights for disease diagnosis and treatment. However, forensic medicine, especially forensic pathology, has witnessed relatively limited progress in the application of molecular biology technologies. A significant challenge in employing molecular techniques for forensic diagnoses lies in the quantitative and qualitative changes observed in diagnostic markers due to sample degradation-a recognized and formidable obstacle. Inspired by the success of DNA sequencing in forensic practices, which enables accurate individual identification even in cases involving degraded and deteriorated tissues and organs, we propose the application of the assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify targets at the transcriptional onset, exploring chromatin and DNA-level alterations for injury and disease inference in forensic samples. This study employs ATAC-seq to explore alterations in chromatin accessibility post-injury and their subsequent changes over a 2-h degradation period, employing traumatic brain injury (TBI) as a representative model. Our findings reveal high sensitivity of chromatin accessibility sites to injury, evidenced by shifts in thousands of peak positions post-TBI. Remarkably, these alterations remain largely unaffected by early degradation. Our results robustly endorse the notion that integrating and incorporating these specific loci for injury and disease diagnosis in forensic samples holds tremendous promise for practical application. We further validated the above results using human cortical tissue, which supported that early degradation did not significantly affect chromatin accessibility. This pioneering advancement in molecular diagnostic techniques may revolutionize the field of forensic science, especially forensic pathology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops 1,2 . Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes 3-5 . Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type 6-9 . Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria 10-12 , we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes., (© 2024. The Author(s).)

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6 μm long and 0.5-0.6 μm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6 days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

SUMMARY In Gram-negative bacteria, the insertion sequence IS 26 is highly active in disseminating antibiotic resistance genes. IS 26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS 26 can also enhance expression of adjacent potential resistance genes. IS 26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS 26 . TU formed by homologous recombination between the bounding IS 26 s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS 26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS 26 is the best characterized IS in the IS 26 family, which includes IS 257 /IS 431 , ISSau10, IS 1216 , IS 1006 , and IS 1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens., Competing Interests: The authors declare no conflict of interest.

Electroactive bacteria, exemplified by Shewanella oneidensis MR-1, have garnered significant attention due to their unique extracellular electron-transfer (EET) capabilities, which are crucial for energy recovery and pollutant conversion. However, the practical application of MR-1 is constrained by its EET efficiency, a key limiting factor, due to the complexity of research methodologies and the challenges associated with the practical use of gene editing tools. To address this challenge, a novel gene integration system, INTEGRATE, was developed, utilizing CRISPR-mediated transposase technologies for precise genomic insertion within the S. oneidensis MR-1 genome. This system facilitated the insertion of extensive gene segments at different sites of the Shewanella genome with an efficiency approaching 100%. The inserted cargo genes could be kept stable on the genome after continuous cultivation. The enhancement of the organism's EET efficiency was realized through two primary strategies: the integration of the phenazine-1-carboxylic acid synthesis gene cluster to augment EET efficiency and the targeted disruption of the SO3350 gene to promote anodic biofilm development. Collectively, our findings highlight the potential of utilizing the INTEGRATE system for strategic genomic alterations, presenting a synergistic approach to augment the functionality of electroactive bacteria within bioelectrochemical systems.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements., Competing Interests: Declaration of interests Cornell University has filed patent applications with J.E.P. and M.T.P. as inventors involving CRISPR-Cas systems associated with transposons that are not related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

IS1111 and IS110 insertion sequence (IS) family members encode an unusual DEDD transposase type and exhibit specific target site selection. The IS1111 group include identifiable subterminal inverted repeats (sTIR) not found in the IS110 type 1 . IS in both families include a noncoding region (NCR) of significant length and, as each individual IS or group of closely related IS selects a different site, we had previously proposed that an NCR-derived RNA was involved in target selection 2 . Here, we find that the NCR is usually downstream of the transposase gene in IS1111 family IS and upstream in the IS110 type. Four IS1111 and one IS110 family members that target different sequences are used to demonstrate that the NCR determines a short seeker RNA (seekRNA) that co-purified with the transposase. The seekRNA is essential for transposition of the IS or a cargo flanked by IS ends from and to the preferred target. Short sequences matching both top and bottom strands of the target are present in the seekRNA but their order in IS1111 and IS110 family IS is reversed. Reprogramming the seekRNA and donor flank to target a different site is demonstrated, indicating future biotechnological potential for these systems., (© 2024. The Author(s).)

Background: A vital step in analyzing single-cell data is ascertaining which cell types are present in a dataset, and at what abundance. In many diseases, the proportions of varying cell types can have important implications for health and prognosis. Most approaches for cell type annotation have centered around cell typing for single-cell RNA-sequencing (scRNA-seq) and have had promising success. However, reliable methods are lacking for many other single-cell modalities such as single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), which quantifies the extent to which genes of interest in each cell are epigenetically "open" for expression., Results: To leverage the informative potential of scATAC-seq data, we developed CAMML with the integration of chromatin accessibility (CAraCAl), a bioinformatic method that performs cell typing on scATAC-seq data. CAraCAl performs cell typing by scoring each cell for its enrichment of cell type-specific gene sets. These gene sets are composed of the most upregulated or downregulated genes present in each cell type according to projected gene activity., Conclusions: We found that CAraCAl does not improve performance beyond CAMML when scRNA-seq is present, but if only scATAC-seq is available, CAraCAl performs cell typing relatively successfully. As such, we also discuss best practices for cell typing and the strengths and weaknesses of various cell annotation options., (© 2024. The Author(s).)

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition., Competing Interests: Declaration of interests X.P., B.B., T.Z., J.R.L., K.T.W., K.W., K.M.H., D.S., and N.O. are all employees of Bio-Techne. N.O. holds stock options in Bio-Techne. B.J.J. was previously an employee of Bio-Techne during the drafting of this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recombination-activating gene 1 (RAG1) is a vital player in V(D)J recombination, a fundamental process in primary B cell and T cell receptor diversification of the adaptive immune system. Current vertebrate RAG evolved from RAG transposon; however, it has been modified to play a crucial role in the adaptive system instead of being irreversibly silenced by CpG methylation. By interrogating a range of publicly available datasets, the current study investigated whether RAG1 has retained a disproportionate level of its original CpG dinucleotides compared to other genes, thereby rendering it more exposed to methylation-mediated mutation. Here, we show that 57.57% of RAG1 pathogenic mutations and 51.6% of RAG1 disease-causing mutations were associated with CpG methylation, a percentage that was significantly higher than that of its RAG2 cofactor alongside the whole genome. The CpG scores and densities for all RAG ancestors suggested that RAG transposon was CpG denser. The percentage of the ancestral CpG of RAG1 and RAG2 were 6% and 4.2%, respectively, with no preference towards CG containing codons. Furthermore, CpG loci of RAG1 in sperms were significantly higher methylated than that of RAG2. In conclusion, RAG1 has been exposed to CpG mediated methylation mutagenesis more than RAG2 and the whole genome, presumably due to its late entry to the genome later with an initially higher CpG content., (© 2024. The Author(s).)

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols., (© 2023. The Author(s).)

Transposable elements engineered to generate random gene fusions in the bacterial chromosome are valuable tools in the study of gene expression. In this protocol, we describe the use of a new series of transposons designed to obtain random fusions to either the lacZY operon or the gene for superfolder green fluorescent protein (sfGFP). Transposition is achieved through the activity of the hyperactive variant of Tn5 transposase (Tnp) whose gene is positioned in cis with respect to the transposable module and under the control of the anyhydrotetracycline (AHTc)-inducible P tet promoter. The transposable module comprises a promoter-less lacZY operon or the sfGFP gene, with or without the lacZ or sfGFP ribosome-binding site, plus a kan gene for selection. The transposon-transposase unit is harbored on an R6K-based suicide plasmid. The plasmid is introduced into recipient cells by electro-transformation and the synthesis of Tn5 Tnp is induced transiently by including AHTc in the recovery medium. Cells are then plated on kanamycin-supplemented medium (without AHTc) where the plasmid DNA is lost and only cells in which transposition has occurred can form colonies. Fusions are detected by screening for colony color on lactose indicator plates ( lacZ transposition) or monitoring green fluorescence ( sfGFP transposition). Depending on whether the reporter gene carries or lacks the ribosome binding sequence, the fusions obtained will either be transcriptional or translational. Parallel screening of colonies grown in the absence and presence of a drug (or condition) eliciting a global regulatory response allows identification of fusions specifically activated or repressed as part of such response., (© 2024 Cold Spring Harbor Laboratory Press.)

Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation. However, these techniques require complex experimental procedures and millions of cells per experiment, which limits their widespread application in life science research. Here, we develop a novel method, Hi-Tag, that identifies high-resolution, long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation (with a phosphorothioate-modified linker). Hi-Tag can be implemented using as few as 100,000 cells, involving simple experimental procedures that can be completed within 1.5 days. Meanwhile, Hi-Tag is capable of using its own data to identify the binding sites of specific proteins, based on which, it can acquire accurate interaction information. Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies, particularly in the context of limited cell availability., (© 2024. Science China Press.)

Glioma is the most common type of primary intracranial malignant tumor, and because of its high invasiveness and recurrence, its prognosis remains poor. The present study investigated the biological function of piggyBac transportable element derived 5 (PGBD5) in glioma. Glioma and para-cancerous tissues were obtained from five patients. Reverse transcription-quantitative PCR and western blotting were used to detect the expression levels of PGBD5. Transwell assay and flow cytometry were used to evaluate cell migration, invasion, apoptosis and cell cycle distribution. In addition, a nude mouse tumor transplantation model was established to study the downstream pathways of PGBD5 and the molecular mechanism was analyzed using transcriptome sequencing. The mRNA and protein expression levels of PGBD5 were increased in glioma tissues and cells. Notably, knockdown of PGBD5 in vitro could inhibit the migration and invasion of glioma cells. In addition, the knockdown of PGBD5 expression promoted apoptosis and caused cell cycle arrest in the G 2 /M phase, thus inhibiting cell proliferation. Furthermore, in vivo experiments revealed that knockdown of PGBD5 expression could inhibit Ki67 expression and slow tumor growth. Changes in PGBD5 expression were also shown to be closely related to the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In conclusion, interference with PGBD5 could inhibit the malignant progression of glioma through the PPAR pathway, suggesting that PGBD5 may be a potential molecular target of glioma.

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., (© 2024 Wiley Periodicals LLC.)

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems., (© 2024. The Author(s).)

The single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) technology provides insight into gene regulation and epigenetic heterogeneity at single-cell resolution, but cell annotation from scATAC-seq remains challenging due to high dimensionality and extreme sparsity within the data. Existing cell annotation methods mostly focus on the cell peak matrix without fully utilizing the underlying genomic sequence. Here we propose a method, SANGO, for accurate single-cell annotation by integrating genome sequences around the accessibility peaks within scATAC data. The genome sequences of peaks are encoded into low-dimensional embeddings, and then iteratively used to reconstruct the peak statistics of cells through a fully connected network. The learned weights are considered as regulatory modes to represent cells, and utilized to align the query cells and the annotated cells in the reference data through a graph transformer network for cell annotations. SANGO was demonstrated to consistently outperform competing methods on 55 paired scATAC-seq datasets across samples, platforms and tissues. SANGO was also shown to be able to detect unknown tumor cells through attention edge weights learned by the graph transformer. Moreover, from the annotated cells, we found cell-type-specific peaks that provide functional insights/biological signals through expression enrichment analysis, cis-regulatory chromatin interaction analysis and motif enrichment analysis., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn 6677 , encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.

Multidrug-resistant Enterobacteriaceae , a prominent family of gram-negative pathogenic bacteria, causes a wide range of severe diseases. Strains carrying the mobile colistin resistance ( mcr-1 ) gene show resistance to polymyxin, the last line of defense against multidrug-resistant gram-negative bacteria. However, the transmission of mcr-1 is not well understood. In this study, genomes of mcr-1 -positive strains were obtained from the NCBI database, revealing their widespread distribution in China. We also showed that IS Apl1 , a crucial factor in mcr-1 transmission, is capable of self-transposition. Moreover, the self-cyclization of IS Apl1 is mediated by its own encoded transposase. The electrophoretic mobility shift assay experiment validated that the transposase can bind to the inverted repeats (IRs) on both ends, facilitating the cyclization of IS Apl1 . Through knockout or shortening of IRs at both ends of IS Apl1 , we demonstrated that the cyclization of IS Apl1 is dependent on the sequences of the IRs at both ends. Simultaneously, altering the ATCG content of the bases at both ends of IS Apl1 can impact the excision rate by modifying the binding ability between IRs and ISAPL1. Finally, we showed that heat-unstable nucleoid protein (HU) can inhibit IS Apl1 transposition by binding to the IRs and preventing ISAPL1 binding and expression. In conclusion, the regulation of IS Apl1 -self-circling is predominantly controlled by the inverted repeat (IR) sequence and the HU protein. This molecular mechanism deepens our comprehension of mcr-1 dissemination., Competing Interests: The authors declare no conflict of interest.

Chromosome rearrangement by LoxP-mediated evolution has emerged as a powerful approach to studying how chromosome architecture impacts phenotypes. However, it relies on the in vitro synthesis of artificial chromosomes. The recently reported CRISPR-associated transposases (CASTs) held great promise for the efficient insertion of abundant LoxP sites directly onto the genome of wild-type strains. In this study, with the fastest-growing bacterium Vibrio natrigens (V. natriegens) as an object, a multiplex genome integration tool derived from CASTs was employed to achieve the insertion of cargo genes at eight specific genomic loci within 2 days. Next, we introduced 30 LoxP sites onto chromosome 2 (Chr2) of V. natriegens. Rigorously induced Cre recombinase was used to demonstrate Chromosome Rearrangement and Modification by LoxP-mediated Evolution (CRaMbLE). Growth characterization and genome sequencing showed that the ~358 kb fragment on Chr2 was accountable for the rapid growth of V. natriegens. The enabling tools we developed can help identify genomic regions that influence the rapid growth of V. natriegens without a prior understanding of genome mechanisms. This groundbreaking demonstration may also be extended to other organisms such as Escherichia coli, Pseudomonas putida, Bacillus subtilis, and so on., (© 2023 Wiley Periodicals LLC.)

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1-2 weeks using available plasmid constructs and standard molecular biology techniques., (© 2024. Springer Nature Limited.)

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future., (© 2024. The Author(s).)

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3'-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase., (© 2023 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The Single-cell Assay for Transposase-Accessible Chromatin with high throughput sequencing (scATAC-seq) has gained increasing popularity in recent years, allowing for chromatin accessibility to be deciphered and gene regulatory networks (GRNs) to be inferred at single-cell resolution. This cutting-edge technology now enables the genome-wide profiling of chromatin accessibility at the cellular level and the capturing of cell-type-specific cis-regulatory elements (CREs) that are masked by cellular heterogeneity in bulk assays. Additionally, it can also facilitate the identification of rare and new cell types based on differences in chromatin accessibility and the charting of cellular developmental trajectories within lineage-related cell clusters. Due to technical challenges and limitations, the data generated from scATAC-seq exhibit unique features, often characterized by high sparsity and noise, even within the same cell type. To address these challenges, various bioinformatic tools have been developed. Furthermore, the application of scATAC-seq in plant science is still in its infancy, with most research focusing on root tissues and model plant species. In this review, we provide an overview of recent progress in scATAC-seq and its application across various fields. We first conduct scATAC-seq in plant science. Next, we highlight the current challenges of scATAC-seq in plant science and major strategies for cell type annotation. Finally, we outline several future directions to exploit scATAC-seq technologies to address critical challenges in plant science, ranging from plant ENCODE(The Encyclopedia of DNA Elements) project construction to GRN inference, to deepen our understanding of the roles of CREs in plant biology.

CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the generation of CHO cell pools using the piggyBac transposon system for mediating gene integration. The method describes the co-transfection of cells with the donor plasmid (coding for the gene of interest) and the helper plasmid (coding for the transposase) using polyethyleneimine (PEI). This is followed by a genetic selection for the generation of a cell pool. The resulting cell pool can be used to start a batch or fed-batch culture. Alternatively, it can be used for generation of clonal cell lines or generation of cell banks for future use., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The Assay for Transposase Accessible Chromatin (ATAC)-seq protocol is optimized to generate global maps of accessible chromatin using limited cell inputs. The Tn5 transposase tagmentation reaction simultaneously fragments and tags the accessible DNA with Illumina Nextera sequencing adapters. Fragmented and adapter tagged DNA is then purified and PCR amplified with dual indexing primers to generate a size-specific sequencing library. The One-Step workflow below outlines the Tn5 nuclei transposition from a range of cell inputs followed by PCR amplification to generate a sequencing library., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Drought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far., Results: In the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes., Conclusions: These results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information., (© 2023. The Author(s).)

Background: Correlation metrics are widely utilized in genomics analysis and often implemented with little regard to assumptions of normality, homoscedasticity, and independence of values. This is especially true when comparing values between replicated sequencing experiments that probe chromatin accessibility, such as assays for transposase-accessible chromatin via sequencing (ATAC-seq). Such data can possess several regions across the human genome with little to no sequencing depth and are thus non-normal with a large portion of zero values. Despite distributed use in the epigenomics field, few studies have evaluated and benchmarked how correlation and association statistics behave across ATAC-seq experiments with known differences or the effects of removing specific outliers from the data. Here, we developed a computational simulation of ATAC-seq data to elucidate the behavior of correlation statistics and to compare their accuracy under set conditions of reproducibility., Results: Using these simulations, we monitored the behavior of several correlation statistics, including the Pearson's R and Spearman's [Formula: see text] coefficients as well as Kendall's [Formula: see text] and Top-Down correlation. We also test the behavior of association measures, including the coefficient of determination R[Formula: see text], Kendall's W, and normalized mutual information. Our experiments reveal an insensitivity of most statistics, including Spearman's [Formula: see text], Kendall's [Formula: see text], and Kendall's W, to increasing differences between simulated ATAC-seq replicates. The removal of co-zeros (regions lacking mapped sequenced reads) between simulated experiments greatly improves the estimates of correlation and association. After removing co-zeros, the R[Formula: see text] coefficient and normalized mutual information display the best performance, having a closer one-to-one relationship with the known portion of shared, enhanced loci between simulated replicates. When comparing values between experimental ATAC-seq data using a random forest model, mutual information best predicts ATAC-seq replicate relationships., Conclusions: Collectively, this study demonstrates how measures of correlation and association can behave in epigenomics experiments. We provide improved strategies for quantifying relationships in these increasingly prevalent and important chromatin accessibility assays., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

DNA transposons play a crucial role in determining the size and structure of eukaryotic genomes. In this study, a new family of IS630-Tc1-mariner (ITm) DNA transposons, named Hiker (HK), was identified. HK is characterized by a DD35E catalytic domain and is distinct from all previously known families of the ITm group. Phylogenetic analyses showed that DD35E/Hiker forms a monophyletic clade with DD34E/Gambol, indicating that they may represent a separate superfamily of ITm. A total of 178 Hiker species were identified, with 170 found mainly in Actinopterygii, one in Chondrichthyes, six in Anura and one in Mollusca. Gambol (GM), on the other hand, are found in invertebrates, with 18 in Arthropoda and one in Platyhelminthes. Hiker transposons have a total length ranging from 2.14 to 3.67 kb and contain a single open reading frame that encodes a protein of approximately 370 amino acids (range 311-413 aa). They are flanked by short terminal inverted repeats (TIRs) of 16-30 base pairs and two base pair (TA) target-site duplications. In contrast, most transposons of the Gambol family have a total length of 1.35-5.96 kb, encode a transposase protein of approximately 350 amino acids (range 306-374 aa), and are flanked by TIRs that range from 32 to 1097 bp in length. Both Hiker and Gambol transposases have several conserved motifs, including helix-turn-helix (HTH) motifs and a DDE domain. Our study observed multiple amplification waves and repeated horizontal transfer (HT) events of HK transposons in vertebrate genomes, indicating their role in diversifying and shaping the genomes of Actinopterygii, Chondrichthyes, and Anura. Conversely, GM transposons showed few Horizontal transfer events. According to cell-based transposition assays, most HK transposons are likely inactive due to the truncated DNA binding domains of their transposases. We present an updated classification of the ITm group based on these findings, which will enhance the understanding of both the evolution of ITm transposons and that of their hosts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)