Integration Events Research Articles

Many evolutionary roads led to virus domestication in ichneumonoid parasitoid wasps.

The investigation of endogenous viral elements (EVEs) has historically focused on only a few lineages of parasitoid wasps, with negative results consistently underreported. Recent studies show that multiple viral lineages were integrated in at least seven instances in Ichneumonoidea and may be much more widespread than previously thought. Increasingly affordable genomic and bioinformatic approaches have made it feasible to search for viral sequences within wasp genomes, opening an extremely promising research avenue. Advances in wasp phylogenetics have shed light on the evolutionary history of EVE integration, although many questions remain. Phylogenetic proximity can be used as a guide to facilitate targeted screening, to estimate the number and age of integration events and to identify taxa involved in major host switches.

Target lines for recombinase-mediated gene stacking in soybean.

Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118 Agrobacterium-mediated transgenic plants that were positive for gus expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a gfp-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a DsRed-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.

A point mutation in HIV-1 integrase redirects proviral integration into centromeric repeats

Retroviruses utilize the viral integrase (IN) protein to integrate a DNA copy of their genome into host chromosomal DNA. HIV-1 integration sites are highly biased towards actively transcribed genes, likely mediated by binding of the IN protein to specific host factors, particularly LEDGF, located at these gene regions. We here report a substantial redirection of integration site distribution induced by a single point mutation in HIV-1 IN. Viruses carrying the K258R IN mutation exhibit a high frequency of integrations into centromeric alpha satellite repeat sequences, as assessed by deep sequencing, a more than 10-fold increase over wild-type. Quantitative PCR and in situ immunofluorescence assays confirm this bias of the K258R mutant virus for integration into centromeric DNA. Immunoprecipitation studies identify host factors binding to IN that may account for the observed bias for integration into centromeres. Centromeric integration events are known to be enriched in the latent reservoir of infected memory T cells, as well as in elite controllers who limit viral replication without intervention. The K258R point mutation in HIV-1 IN is also present in databases of latent proviruses found in patients, and may reflect an unappreciated aspect of the establishment of viral latency.

Vertical Integration and Foreclosure: Evidence from Production Network Data

This paper studies the prevalence of potential anticompetitive effects of vertical mergers using a novel data set on U.S. and international buyer-seller relationships and across a large range of industries. We find that relationships are more likely to break when suppliers vertically integrate with one of the buyers’ competitors than when they vertically integrate with an unrelated firm. This relationship holds for both domestic and cross-border mergers and for domestic and international relationships. It also holds when instrumenting mergers using exogenous downward pressure on the supplier’s stock prices, suggesting that reverse causality is unlikely to explain the result. In contrast, the relationship vanishes when using rumored or announced but not completed integration events. Firms experience a substantial drop in sales when one of their suppliers integrates with one of their competitors. This sales drop is mitigated if the firm has alternative suppliers in place. These findings are consistent with anticompetitive effects of vertical mergers, such as vertical foreclosure, rising input costs for rivals, or self-foreclosure. This paper was accepted by Joshua Gans, business strategy. Funding: This work was supported by a Banque de France/Sciences Po Partnership. Supplemental Material: The data files and online appendices are available at https://doi.org/10.1287/mnsc.2022.4363 .

Construction Progress and Aviation Flight Test of BDSBAS

As an important part of the BeiDou global navigation satellite system (BDS-3), the BeiDou satellite-based augmentation system (BDSBAS) aims to provide a high-integrity navigation service for users in China and its surrounding areas. At present, BDSBAS has completed the preliminary construction of the system and formed the single frequency (SF) augmentation service. The paper introduces the composition and working process of BDSBAS, and expounds the solution and performance evaluation methods of the BDSBAS SF service. Tests based on a general aviation aircraft were carried out at Caihu Airport in Shenyang, China, which simulated the aircraft’s take-off and landing operation during the approach phase and the transition flight operation during the en-route phase. On this basis, the BDSBAS SF service performance was analyzed and evaluated. The horizontal positioning errors of the three flight tests are 1.62, 1.27, and 1.49 m, respectively, and the vertical positioning errors are 4.38, 4.30, and 4.04 m, respectively. In the first two tests, the APV-I availability and continuity of the service reach 100%. In the last test, a receiver false alarm event occurred, resulting in an APV-I availability and continuity of 99.993% and 99.989%, respectively. The event was confirmed to be caused by some aviation environmental factors and was not related to the system service. No integrity events occurred during the tests. The test results show that the positioning accuracy, availability, and integrity of the BDSBAS SF service meet the APV-I level requirements of the ICAO standards, which preliminarily illustrates the ability of BDSBAS to be used in the field of civil aviation.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Background & AimsHBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.MethodsDNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.ResultsHBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.ConclusionsTargeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Lay summaryFresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Recombinase-mediated gene stacking in cotton.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv. Coker 312) target lines suitable for Mycobacteriophage Bxb1 recombinase-mediated gene stacking. Obtained through the empirical screening of random insertion events, each of these target lines contains a single intact copy of the target construct with precise sequences of RS2, lox, and attP sites that is not inserted within or close to a known gene or near a centromere and shows good expression of the reporter gene gfp. Gene stacking was tested with insertion of different combinations of three candidate genes for resistance to verticillium wilt into three cotton target lines: CTS1, CTS3, and CTS4. Nine site-specific integration events were recovered from 95 independently transformed embryogenic calluses. Southern and DNA sequence analyses of regenerated plants confirmed precise site-specific integration, and resistance to verticillium wilt was observed for plant CTS1i3, which has a single precise copy of site-specifically integrated DNA. These cotton target lines can serve as foundation lines for recombinase-mediated gene stacking to facilitate precise DNA integration and introgression to field cultivars.

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR

Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.

InterTADs: integration of multi-omics data on topologically associated domains, application to chronic lymphocytic leukemia.

ABSTRACTThe integration of multi-omics data can greatly facilitate the advancement of research in Life Sciences by highlighting new interactions. However, there is currently no widespread procedure for meaningful multi-omics data integration. Here, we present a robust framework, called InterTADs, for integrating multi-omics data derived from the same sample, and considering the chromatin configuration of the genome, i.e. the topologically associating domains (TADs). Following the integration process, statistical analysis highlights the differences between the groups of interest (normal versus cancer cells) relating to (i) independent and (ii) integrated events through TADs. Finally, enrichment analysis using KEGG database, Gene Ontology and transcription factor binding sites and visualization approaches are available. We applied InterTADs to multi-omics datasets from 135 patients with chronic lymphocytic leukemia (CLL) and found that the integration through TADs resulted in a dramatic reduction of heterogeneity compared to individual events. Significant differences for individual events and on TADs level were identified between patients differing in the somatic hypermutation status of the clonotypic immunoglobulin genes, the core biological stratifier in CLL, attesting to the biomedical relevance of InterTADs. In conclusion, our approach suggests a new perspective towards analyzing multi-omics data, by offering reasonable execution time, biological benchmarking and potentially contributing to pattern discovery through TADs.

Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation

Hepatocellular carcinogenesis of hepatitis B virus (HBV) infection may arise from integration of viral DNA into the host genome. We aimed to gauge the effect of viral inhibition on transcriptionally active HBV-host integration events and explore the correlation of viral integrations with host gene dysregulation. We leveraged data and biospecimens from an interventional trial, in which patients with HBV viremia above 2000 IU/mL and minimally raised serum liver enzyme were randomized to receive tenofovir disoproxil fumarate (TDF) or placebo for 3 years. Total RNA-sequencing was performed on paired liver biopsies taken before and after the 3-year intervention in 119 patients. Virus-host chimeric reads were captured to quantify the number of distinct viral integrations. Dysregulation of a host gene disrupted by viral integration was defined by aberrant expression >2 standard deviations away from samples without viral integration. The TDF (n= 64) and placebo groups (n= 55) were comparable at baseline. Expressed viral integrations were detected in all pre- and posttreatment samples. The number of distinct viral integrations significantly correlated with circulatory biomarkers indicative of viral activities including HBV DNA, RNA, and viral antigens (P < .0003 for all correlations). Moreover, TDF vs placebo achieved a significantly greater reduction in distinct viral integrations, with 3.28-fold and 1.81-fold decreases in the expressed integrations per million reads, respectively (analysis of covariance, P= .037). Besides, viral integrations significantly correlated with host gene dysregulation. Inhibition of viral replication reduces the number of transcriptionally active distinct HBV-host DNA integrations in patients with substantial viremia. Given the mutagenic potentials of viral integrations, such treatment effects should be considered in patient management.

Współpraca miejskich i wiejskich szkół ogólnodostępnych z placówkami specjalnymi

Introduction: Improving the quality of inclusive education is determined by many factors. One of them, applied in many countries, is the support for mainstream schools by building a partnership with special schools and by making use of their resources. The article is focused on the issue of cooperation between mainstream schools and special ones, viewed from the standpoint of the staff of the former. What has been additionally undertaken is the examination of this partnership as regards the (rural and urban) environment of mainstream school. Research Aim: The studies were aimed at recognizing the forms of collaboration between mainstream and special schools comprised in the declarations of mainstream school staff, as well as specifying the relationship between the intensity of collaboration indications and the (rural and urban) location of mainstream schools. Method: The studies were conducted with the use of diagnostic poll method. The data were collected with the use of a questionnaire for the staff of mainstream primary schools. For the needs of this article, the issue was analysed of good practices applied to build the inclusive culture of school. 2548 answers were obtained (1498 from rural and 1046 from urban schools, in 4 cases the location was not provided so these facilities were excluded from the research). From all the answers, the declarations were selected which concerned the cooperation of mainstream schools with special ones. Results: An analysis of the data revealed a meagre level of cooperation. Only 2.01% of urban and 1.87% of rural schools declared different forms of collaboration with special facilities. They mostly concerned the common organizing of integration events. The (rural / urban) environment practically does not differentiate the level of cooperation. Conclusions: The building of cooperation (which, in the light of the analysed expert literature, is beneficial to both types of school) requires some systemic solutions that initiate various forms of partnership and the creation of a set of stimuli (e.g. in the form of projects) encouraging to regular cooperation.

Isling: A Tool for Detecting Integration of Wild-Type Viruses and Clinical Vectors

Detecting viral and vector integration events is a key step when investigating interactions between viral and host genomes. This is relevant in several fields, including virology, cancer research and gene therapy. For example, investigating integrations of wild-type viruses such as human papillomavirus and hepatitis B virus has proven to be crucial for understanding the role of these integrations in cancer. Furthermore, identifying the extent of vector integration is vital for determining the potential for genotoxicity in gene therapies. To address these questions, we developed isling, the first tool specifically designed for identifying viral integrations in both wild-type and vector from next-generation sequencing data. Isling addresses complexities in integration behaviour including integration of fragmented genomes and integration junctions with ambiguous locations in a host or vector genome, and can also flag possible vector recombinations. We show that isling is up to 1.6-fold faster and up to 170% more accurate than other viral integration tools, and performs well on both simulated and real datasets. Isling is therefore an efficient and application-agnostic tool that will enable a broad range of investigations into viral and vector integration. These include comparisons between integrations of wild-type viruses and gene therapy vectors, as well as assessing the genotoxicity of vectors and understanding the role of viruses in cancer.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an “integrated standard”; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Staphylococcal Phages Adapt to New Hosts by Extensive Attachment Site Variability.

ABSTRACTBacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health.

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach.

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

Comprehensive characterization of viral integrations and genomic aberrations in HBV-infected intrahepatic cholangiocarcinomas.

Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors. We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n=167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n=59), and conventional (n=154) and fluke-related iCCAs (n=16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA. Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

The integration model of hepatitis B virus genome in hepatocellular carcinoma cells based on high-throughput long-read sequencing

HBV integration and function has gradually been expanding. However, the exact mode of HBV integration remains unclear. In our research, the high-throughput long-read sequencing was combined with bioinformatics to study the complete mode of HBV integration in hepatocellular carcinoma (HCC) cells. The results demonstrated that: 1) The HBV insertion sequences of HBV integration events accounted for 49.5% of the total HBV sequences. 2) Short insertion segments with the length of 0–1 kbp accounted for 50% and the long insertion segments (>3 kbp) accounted for 25% of HBV insertion events. 3)There were different HBV insertion length in the breakpoints formed within different regions. 4) The occurrence of HBV integration events was accompanied by more frequent structural variations. 5)Furthermore, multiple HBV integration patterns were confirmed based on complete HBV insertion sequences. Our research not only clarified a variety of perfect HBV integration models but also determined multiple specific features of HBV integration.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

BackgroundRedirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging.MethodsIn the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells.ResultsWe detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells.ConclusionLooking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

SUMMARY Agrobacterium tumefaciens‐mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T‐DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)‐mediated end joining (TMEJ). Targeting of the T‐DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T‐DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ‐proficient plants we predominantly found GT events accompanied by random T‐DNA integrations, GT events obtained in the teb mutant background lacked additional T‐DNA copies, corroborating the essential role of Polθ in T‐DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high‐quality GT events in crop plants.

Genomic Characterization of a Proteus sp. Strain of Animal Origin Co-Carrying blaNDM-1 and lnu(G).

The emergence of carbapenem-resistant Proteus represents a serious threat to global public health due to limited antibiotic treatment options. Here, we characterize a Proteus isolate NMG38-2 of swine origin that exhibits extensive drug resistance, including carbapenems. Whole-genome sequencing based on Illumina and MinION platforms showed that NMG38-2 contains 24 acquired antibiotic resistance genes and three plasmids, among which, pNDM_NMG38-2, a pPvSC3-like plasmid, is transferable and co-carries blaNDM-1 and lnu(G). Sequence analysis of pPvSC3-like plasmids showed that they share a conserved backbone but have a diverse accessory module with complex chimera structures bearing abundant resistance genes, which are facilitated by transposons and/or homologous recombination. The acquisition of blaNDM-1 in pNDM_NMG38-2 was due to the ISCR1-mediated integration event. Comprehensive analysis of the lnu(G)-bearing cassettes carried by bacterial plasmids or chromosomes revealed a diversification of its genetic contexts, with Tn6260 and ISPst2 elements being the leading contributors to the dissemination of lnu(G) in Enterococcus and Enterobacteriaceae, respectively. In conclusion, this study provides a better understanding of the genetic features of pPvSC3-like plasmids, which represent a novel plasmid group as a vehicle mediating the dissemination of blaNDM-1 among bacteria species. Moreover, our results highlight the central roles of Tn6260 and ISPst2 in the spread of lnu(G).