Large Fragments Research Articles

CRISPR/Cas9 effectively generate chromosome structural variations in rice protoplasts

AbstractChromosome structural variations (SVs), such as deletion, duplication, inversion, and translocation, are important contributors to genetic diversification and crop improvement. Using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas9), desired SVs involving large DNA fragments have been created in rice (Oryza sativa L.), maize (Zea mays L.), and Arabidopsis (Arabidopsis thaliana L.). However, it is still uncertain whether the size of DNA fragment involved could be a prohibiting factor to generate Cas9‐mediated SVs. In this study, we constructed five CRISPR/Cas9 vectors, each expressing two single‐guide RNAs (sgRNAs), to cut two sites spacing at 0.5, 5, 10, 20, and 30 Mb on rice chromosome 4 (Chr4), respectively. Meanwhile, another CRISPR/Cas9 vector cutting two sites, one on Chr4 and the other on Chr1, was also constructed for creation of chromosomal translocation between Chr1 and Chr4. These vectors were transfected into rice protoplasts by polyethylene glycol–mediated transformation. Specific primers were designed to detect desired SV events. The results showed that all designed SVs could be effectively generated by CRISPR/Cas9 in rice protoplasts. This study suggested that the size of DNA fragment involved is unlikely a prohibiting factor for creation of desired SV events.

Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002

Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.

Genome characterization of Rift Valley fever virus isolated from cattle, goats and sheep during interepidemic periods in Kenya

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus of the Phlebovirus genus in the phenuviridae family. Its genome is trisegmented with small (S), medium (M) and large (L) fragments. In nature, the virus exists as a single serotype that is responsible for outbreaks of Rift Valley fever (RVF), a zoonotic disease that often occurs in Africa and the Middle East. RVFV genomes are thought to undergo both recombination and reassortment and investigations of these events is important for monitoring the emergence of virulent strains and understanding the evolutionary characteristics of this virus. The aim of this study was to characterize the genomes of RVFV isolates from cattle, sheep, and goats collected during an interepidemic period in Kenya between June 2016 and November 2021. A total of 691 serum samples from cattle (n = 144), goats (n = 185) and sheep (n = 362) were analysed at the Central Veterinary Laboratories. The competitive IgM-capture ELISA, was used to screen the samples; 205 samples (29.67%) tested positive for RVFV. Of the 205 positive samples, 42 (20.5%) were from cattle, 57 (27.8%) from goats, and 106 (51.7%) from sheep. All the IgM-positive samples were further analyzed by qPCR, and 24 (11.71%) tested positive with Ct values ranging from 14.788 to 38.286. Two samples, 201808HABDVS from sheep and 201810CML3DVS from cattle, had Ct values of less than 20.0 and yielded whole genome sequences with 96.8 and 96.4 coverage, respectively. There was no statistically significant evidence of recombination in any of the three segments and also phylogenetic analysis showed no evidence of reassortment in the two isolated RVFV segments when compared with other isolates of different lineages from previous outbreaks whose genomes are deposited in the GenBank. No evidence of reassortment leaves room for other factors to be the most probable contributors of change in virulence, pathogenicity and emergence of highly virulent strains of the RVFV.

Regional active transcription associates with homoeologous exchange breakpoints in synthetic Brassica tetraploids.

Polyploidization plays a crucial role in plant evolution and is becoming increasingly important in breeding. Structural variations and epigenomic repatterning have been observed in synthetic polyploidizations. However, the mechanisms underlying the occurrence and their effects on gene expression and phenotype remain unknown. Here, we investigated genome-wide large deletion/duplication regions (DelDups) and genomic methylation dynamics in leaf organs of progeny from the first eight generations of synthetic tetraploids derived from Chinese cabbage (Brassica rapa L. ssp. pekinensis) and cabbage (Brassica oleracea L. var. capitata). One- or two-copy DelDups, with a mean size of 5.70 Mb (400 kb to 65.85 Mb), occurred from the first generation of selfing and thereafter. The duplication of a fragment in one subgenome consistently coincided with the deletion of its syntenic fragment in the other subgenome, and vice versa, indicating that these DelDups were generated by homoeologous exchanges (HEs). Interestingly, the larger the genomic syntenic region, the higher the frequency of DelDups, further suggesting that the pairing of large homoeologous fragments is crucial for HEs. Moreover, we found that the active transcription of continuously distributed genes in local regions is positively associated with the occurrence of HE breakpoints. In addition, the expression of genes within DelDups exhibited a dosage effect, and plants with extra parental genomic fragments generally displayed phenotypes biased toward the corresponding parent. Genome-wide methylation fluctuated remarkably, which did not clearly affect gene expression on a large scale. Our findings provide insights into the early evolution of polyploid genomes, offering valuable knowledge for polyploidization-based breeding.

CILF: CRISPR/Cas9 based integration of large DNA fragments in Saccharomyces cerevisiae.

Genome integration technology has markedly expedited the construction of cell factories. However, its application is currently limited by the inefficient integration of large DNA fragments. Here, we report a CRISPR/Cas9 based integration of large DNA fragments (CILF) method to efficiently integrate large DNA fragments in Saccharomyces cerevisiae. In this approach, a fusion protein, Cas9-Brex27-FadR, was employed for the targeted delivery of donor plasmid to double-strand breaks (DSBs), while simultaneously recruiting Rad51 to enhance the efficiency of homologous recombination (HR). Our findings demonstrate that this method can achieve an integration efficiency of 98% for 10 kb DNA fragments and nearly 80% for 40 kb DNA fragments at a single site, using donor plasmids with 1000 bp homology arms (HAs) and 12 FadR binding sites (BSs). The CILF technique significantly enriches the synthetic biology toolbox of S. cerevisiae, offering significant potential to propel advancements in both synthetic biology and metabolic engineering.

Comparative transcriptome analysis of Cyperus esculentus and C. rotundus with contrasting oil contents in tubers defines genes and regulatory networks involved in oil accumulation

Plant vegetative organs present great potential for lipid storage, with tubers of Cyperus esculentus as a unique example. To investigate the genome and transcriptomic features of C. esculentus and related species, we sequenced and assembled the C. esculentus genome at the contig level. Through a comparative study of high-quality transcriptomes across 36 tissues from high-oil and intermediate-oil C. esculentus and low-oil Cyperus rotundus, we identified potential genes and regulatory networks related to tuber oil accumulation. First, we identified tuber-specific genes in two C. esculentus cultivars. Second, genes involved in fatty acid (FA) biosynthesis, triacylglycerol synthesis, and TAG packaging presented increased activity in the later stages of tuber development. Notably, tubers with high oil contents presented higher levels of these genes than those with intermediate oil contents did, whereas tubers with low oil contents presented minimal gene expression. Notably, a large fragment of the FA biosynthesis rate-limiting enzyme-encoding gene BCCP1 was missing from the C. rotundus transcript, which might be responsible for blocking FA biosynthesis in its tubers. WGCNA pinpointed a gene module linked to tuber oil accumulation, with a coexpression network involving the transcription factors WRI1, MYB4, and bHLH68. The ethylene-related genes in this module suggest a role for ethylene signaling in oil accumulation, which is supported by the finding that ethylene (ETH) treatment increases the oil content in C. esculentus tubers. This study identified potential genes and networks associated with tuber oil accumulation in C. esculentus, highlighting the role of specific genes, transcription factors, and ethylene signaling in this process.

Prone position fibula nail and tibia buttress plate for large Volkmann fracture-dislocations technical note and preliminary results on a short case series

Large posterolateral tibial fragments, known as Volkmann fractures, are common in ankle fracture-dislocations and typically require open reduction and fixation with interfragmentary screws and often buttress plates using a prone posterolateral approach. In this setting, fibula plating often necessitates dissection of the lateral window between the peroneal tendons and the skin, increasing wound-related complications. In recent years, intramedullary nailing of fibula fractures has gained popularity as a minimally invasive technique that allows load-sharing stabilization. However, this procedure has traditionally been performed in the supine position, which can pose challenges if a Volkmann-type fracture is present, requiring repositioning and losing access to the fibula through the posterolateral approach. Our objective is to describe a technique for fixing ankle fracture-dislocations with large posterolateral fragments using tibial buttress plates and fibula nails through a prone posterolateral approach without dissecting the lateral window. Additionally, we will present a case series with preliminary results.

Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.

Metabolic responses of the marine mussel Mytilus galloprovincialis after exposure to microplastics of different shapes and sizes

Microplastics (MP) are ubiquitous pollutants with diverse shapes, sizes, and characteristics that pose critical risks to marine organisms and the environment. In this study, we used the Mediterranean mussel Mytilus galloprovincialis as a marine benthic organism model to investigate the metabolic consequences of exposure to different polyethylene terephthalate MP sizes and shapes: round (27–32 μm), small fibers (200–400 μm), large fibers (3000 μm), small fragments (20 μm), medium fragments (45–75 μm), and large fragments (>150 μm). After exposure to high concentrations (100 mg L−1) of MP for 14 days, round and small fiber-type MP were highly accumulated in mussels. Metabolomic analysis revealed that exposure to round and small fiber-type MP induced significant changes in 150 metabolites. Partial least squares-discriminate analysis (PLS-DA) showed that the round and small fiber MP treatment groups displayed similar cluster patterns that differed from those of the control group. In addition, only 22 annotated metabolites related to histidine, valine, leucine, and isoleucine degradation/biosynthesis and vitamin B6 and aminoacyl-tRNA biosynthesis were significantly affected by round or small fiber-type MP. Among the histidine metabolites, round and small fiber-type MP upregulated the levels of L-histidine, L-glutamate, carnosine, imidazole-4-acetaldehyde, 4-imidazolone-5-propanoate, and methylimidazole acetaldehyde and downregulated methylimidazole acetic acid and N-formimino-L-glutamate. These results suggest novel insights into the potential pathways through which MP of specific sizes and shapes affect metabolic processes in mussels.

Enlarging interface reverses the dominance of fungi over bacteria in litter decomposition

Soil microorganisms are primary decomposers driving carbon and nutrient cycling in terrestrial ecosystems. One prevailing view is that fungi, rather than bacteria, play a predominant role in litter decomposition. However, the roles of bacteria and factors that restrict their activity during decomposition remain unclear. We hypothesized that the limiting activity of bacterial decomposers is associated with litter size beyond chemical quality. To address this gap, we conducted a 180-d decomposition microcosm experiment to investigate the effect of fragment size (large, 1–2 mm; middle, 0.18–0.28 mm; small, <0.07 mm) of oak and pine litters on bacterial or fungal decomposition. Bacterial and fungal decomposition were accelerated with fragment size decrease, suggesting that an interface effect existed between microbial decomposers and litter. Generally, the decomposition ability of bacteria was more sensitive to changes in fragment size compared to fungi. Fungi resulted in faster decomposition of large fragments than bacteria. For small oak litter fragments, bacteria showed faster decomposition than fungi, whereas the opposite was true for small pine litter. Therefore, the decomposition dominance of bacteria and fungi was regulated by fragment size and influenced by the chemical quality of litter, especially the lignin:N ratio. The contrasting decomposition dominances of bacteria versus fungi were likely attributed to filamentous fungi penetrating litter interiors and forming mycelial bridges between scattered litters. Bacteria resided on litter surfaces and even formed biofilms. Consistent with the findings of the microcosm experiment, the proportion of small fragments was greater in the fresh litter layer than in the decomposed layer in the field, and greater in the pine forest than in the oak forest, suggesting the fresh, large, and low-quality litter was preferentially fragmented by fungi. Consequently, the dominance of fungi and bacteria during litter decomposition in the conventional view should be revisited considering the litter size.

High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus.

Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Posterior Wall Fragments in Acetabular Both-Column Fractures: Morphology, Type, and the Significance of its Projection.

Most both-column acetabular fractures are combined with posterior wall fragments. However, the morphology of this posterior wall is varied, and how to fix this posterior wall remains a controversial topic. To investigate the morphological characteristics of posterior wall fragments of both-column acetabular fractures and select corresponding fixation methods. Data from 352 patients with acetabular fractures admitted to the level one trauma centre in our hospital between January 2006 and December 2022 were collected. The morphology of posterior wall fragments was observed and analyzed in 83 cases of both-column acetabular fractures and classified according to the consistency of posterior wall morphology. A fracture map of the posterior wall was created on a normal template according to the three morphological types of posterior wall fragments. Finally, the high-incidence area of the posterior wall fracture was projected onto the iliac fossa and the medial side of the posterior column to guide the fixation of the posterior wall fragment using the anterior intrapelvic approach. Fractures were divided into four types: I, large posterior wall fragment which was high in the ilium bone (34 cases, 41.0%); II, posterior wall fragment in the acetabular parietal region (18 cases, 21.7%); III, posterior wall marginal fracture (10 cases, 12.0%); and IV, non-combined posterior wall fracture (21 cases, 25.3%). The most common morphologies of the posterior wall fragments of the first two types were mapped and projected onto the anterior iliac inner plate and medial side of the posterior column, where the corresponding area could be used to guide the insertion of the internal fixation. Both-column acetabular fractures combined with posterior wall fractures can be divided into four types according to the morphology of the posterior wall fragment. Understanding the corresponding three-dimensional morphology and projection position of different types of these fragments can help surgeons determine the position and orientation of internal fixation of posterior wall fractures.

MAX germline mutation-associated pheochromocytoma-paraganglioma syndrome: multiple endocrine neoplasia type 5

The individualized precision management of hereditary pheochromocytoma (PHEO) and paraganglioma (PGL) syndromes (PPGLs) based on molecular diagnosis and molecular subtype is becoming more popular. The newly discovered MAX germline mutation-associated PPGLs are autosomally dominant and rare. To raise awareness and explore the effective management of individual diagnosis and treatment, the relevant literature published between January 2011 and February was systematically reviewed. There were a total of 101 patients in the 77 families, involving all 5 exons, containing 44 types of MAX germline mutations and mostly concentrated in exons 3 and 4 (64.4%), the main mutations were nonsense mutations and missense mutations (72.3%), and some were large fragment deletions or insertions, intron variant, gene fusion mutations were relatively infrequent. Furthermore, about 10% of the patients had a paternal parent-of-origin effect. Among the 101 patients, 96 (95.0%) developed PHEO including 15 metastatic PHEO, 61 bilateral PHEO and 35 unilateral PHEO. The age of diagnosis was (31.7±10.9) years (range: 13 to 80 years). The male to female ratio was 1.2∶1. Eleven were accompanied with chest and abdominal PGL. Eight (7.9%) were accompanied by functional pituitary adenoma. And 12 (11.9%) developed other neuroendocrine tumors (NET), of which 8 were accompanied by PHEO, including 4 hyperparathyroidism, 1 gangliocytoma and neuroblastoma, 1 pancreatic NET, 1 medullary thyroid carcinoma and 1 C cell hyperplasia. Six presented concomitant non-NET, including 1 tongue squamous cell carcinoma, 1 papillary thyroid carcinoma, 1 prostate cancer, 1 renal oncocytoma, 1 breast cancer with renal oncocytoma, and 1 thoracic chondrosarcoma with multifocal adenocarcinoma of lung. The remaining 5 cases (5.0%), including 4 other NET (2 ganglioblastoma, 1 abdominal neuroblastoma and 1 pancreatic NET) and 1 asymptomatic child, did not present PHEO. The MAX germline mutation may cause a novel multiple endocrine neoplasia, which can be described as type 5. A comprehensive baseline assessment of neural crest cell-derived diseases such as PPGL, pituitary adenoma, hyperparathyroidism, and/or gangliocytoma (neuroblastoma) was recommended for all people with MAX germline mutations, and the risk of bilateral and/or metastatic PHEO should also be considered. In contrast, patients with PPGLs combined with other NET, such as functional pituitary adenoma, should undergo genetic testing and pedigree screening that includes at least the MAX gene.

Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of β-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

Effect of the Inter-Ring Delay Time on Rock Fragmentation: Field Tests at the Underground Mine

The effect of inter-ring delay time (IRDT) on rock fragmentation in the tunnel excavation blasting was studied at the Xinjiang Beizhan Iron Mine, China, to improve the rock fragmentation and optimize the blast design. Blasting tests were conducted with an IRDT of 50, 100, 150, 200, and 500 ms; each adjacent ring had an equal IRDT, and two replicate tests were conducted. The blasting plan with IRDT of 100 ms was the original blasting plan used in the mine. Two optimized blasting plans were proposed and implemented based on the experimental results, along with a control experiment using the original blasting plan. The fragment size of each blast test was measured and analyzed by the block-analyzed software. Both collision theoretical and field tests indicated that the IRDT plays an important role in rock fragmentation and that as the IRDT increases, the degree of rock fragmentation increases first and then decreases. For example, the fragment sizes X20, X50, and X80 showed an increase followed by a decrease; the percentage of large fragments (1-P750) showed a decline followed by a rise; the percentage of small fragments P25 showed an increase followed by a decline. The blasting plan with an IRDT of 150 ms had the most optimal rock fragmentation effect, with the lowest percentage of large fragments, the highest percentage of small fragments, and the smallest average fragment size of X50. Furthermore, the two optimized blasting plans demonstrated better control over blasting costs and rock fragmentation compared to the original blasting plan.

Evaluation of artificial intelligence-assisted morphological analysis for platelet count estimation.

This study aims to assess the performance of the platelet count estimation using artificial intelligence technology on the MC-80 digital morphology analyzer. Digital morphology analyzer uses two different computational principles for platelet count estimation: based on PLT/RBC ratio (PLT-M1) and estimate factor (PLT-M2). 977 samples with various platelet counts (low, median, and high) were collected. Out of these, 271 samples were immunoassayed using CD61 and CD41 antibodies. The platelet counts obtained from the hematology analyzer (PLT-I and PLT-O), digital morphology analyzer (PLT-M1 and PLT-M2), and flow cytometry (PLT-IRM) were compared. There was no significant deviation observed before and after verification for both PLT-M1 and PLT-M2 across the analysis range (average bias: -0.845/-0.682, 95% limit of agreement (LOA): -28.675-26.985/-29.420-28.056). When platelet alarms appeared, PLT-M1/PLT-M2 showed the strongest correlation with PLT-IRM than PLT-I with PLT-IRM (r: 0.9814/0.9796 > 0.9601). The correlation between PLT-M1/PLT-M2 and PLT-IRM was strong for samples with interference, such as large platelets or RBC fragments, but relatively weak in small RBCs. The deviation between PLT-M1 and PLT-M2 is related to the number of RBCs. Compared with PLT-I, PLT-M1/PLT-M2 showed higher accuracy for platelet transfusion decisions, especially for samples with low-value PLT. The novel platelet count estimation on the MC-80 digital morphology analyzer provides high accuracy, especially the reviewed result, which can effectively confirm suspicious platelet count.

A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster.

The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.

Establishing a straightforward I-SceI-mediated recombination one-plasmid system for efficient genome editing in P. putida KT2440.

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as a counterselection marker to accelerated plasmid curing; (2) double-strand break introducing gene I-sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

Large Felid Tracks and Bones From Chilly Bowl Cave, Arkansas

Multiple large cat tracks were found within Chilly Bowl Cave, Arkansas, representing at least two individuals of the genus Panthera. Large felid bone fragments were also found in a passageway near the tracks. These poorly preserved skeletal remains primarily consist of metapodials and phalanges and represent the North American Pleistocene jaguar, Panthera onca. Fusion of metapodial epiphyses indicate adult foot size had been reached for this large jaguar. One set of tracks is also consistent with the size and morphology of P. onca. The other tracks are much larger than is known for extant or prehistoric jaguars, and are here attributed to Panthera atrox, the American lion. An alternate hypothesis for the smaller tracks is that they could represent a younger P. atrox instead of P. onca. Regardless of the inability to identify the smaller feline prints to species, these remains and tracks from Chilly Bowl Cave record cave utilization by two separate Pleistocene felids, interpreted here as jaguar and American lion.

Integration of large and diverse angiosperm DNA fragments into Asian Gnetum mitogenomes

BackgroundHorizontal gene transfer (HGT) events have rarely been reported in gymnosperms. Gnetum is a gymnosperm genus comprising 25‒35 species sympatric with angiosperms in West African, South American, and Southeast Asian rainforests. Only a single acquisition of an angiosperm mitochondrial intron has been documented to date in Asian Gnetum mitogenomes. We wanted to develop a more comprehensive understanding of frequency and fragment length distribution of such events as well as their evolutionary history in this genus.ResultsWe sequenced and assembled mitogenomes from five Asian Gnetum species. These genomes vary remarkably in size and foreign DNA content. We identified 15 mitochondrion-derived and five plastid-derived (MTPT) foreign genes. Our phylogenetic analyses strongly indicate that these foreign genes were transferred from diverse eudicots—mostly from the Rubiaceae genus Coptosapelta and ten genera of Malpighiales. This indicates that Asian Gnetum has experienced multiple independent HGT events. Patterns of sequence evolution strongly suggest DNA-mediated transfer between mitochondria as the primary mechanism giving rise to these HGT events. Most Asian Gnetum species are lianas and often entwined with sympatric angiosperms. We therefore propose that close apposition of Gnetum and angiosperm stems presents opportunities for interspecific cell-to-cell contact through friction and wounding, leading to HGT.ConclusionsOur study reveals that multiple HGT events have resulted in massive amounts of angiosperm mitochondrial DNA integrated into Asian Gnetum mitogenomes. Gnetum and its neighboring angiosperms are often entwined with each other, possibly accounting for frequent HGT between these two phylogenetically remote lineages.