Multiple Insertions Research Articles

Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans.

Highly efficient generation of deletions, substitutions, and small insertions (up to ~ 150 bp) into the Caenorhabditis elegans genome by CRISPR/Cas9 has been facilitated by the use of single-stranded oligonucleotide donors as repair templates. However, insertion of larger sequences such as fluorescent markers and other functional domains remains challenging due to uncertainty of optimal performance between single-stranded or double-stranded repair templates and labor-intensive as well as inefficient protocols for their preparations. Here, we simplify the generation of long ssDNA as donors in CRISPR/Cas9. High yields of ssDNA can be rapidly generated using a standard PCR followed by a single enzymatic digest with lambda exonuclease. Comparison of long ssDNA donors obtained using this method to dsDNA demonstrates orders of magnitude increased insertion frequency for ssDNA donors. This can be leveraged to simultaneously generate multiple large insertions as well as successful edits without the use of selection or co-conversion (co-CRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized, and labor-minimal protocol.

Characterizing Planar Tanglegram Layouts and Applications to Edge Insertion Problems

Tanglegrams are formed by taking two rooted binary trees $T$ and $S$ with the same number of leaves and uniquely matching each leaf in $T$ with a leaf in $S$. They are usually represented using layouts that embed the trees and matching in the plane. Given the numerous ways to construct a layout, one problem of interest is the Tanglegram Layout Problem, which is to efficiently find a layout that minimizes the number of crossings. This parallels a similar problem involving drawings of graphs, where a common approach is to insert edges into a planar subgraph. In this paper, we explore inserting edges into a planar tanglegram. Previous results on planar tanglegrams include a Kuratowski Theorem, enumeration, and an algorithm for finding a planar layout. We build on these results and characterize all planar layouts of a planar tanglegram. We then apply this characterization to construct a quadratic-time algorithm that inserts a single edge optimally. Finally, we generalize some results to multiple edge insertion.

Genomic Assessment of the Contribution of the Wolbachia Endosymbiont of Eurosta solidaginis to Gall Induction.

We explored the genome of the Wolbachia strain, wEsol, symbiotic with the plant-gall-inducing fly Eurosta solidaginis with the goal of determining if wEsol contributes to gall induction by its insect host. Gall induction by insects has been hypothesized to involve the secretion of the phytohormones cytokinin and auxin and/or proteinaceous effectors to stimulate cell division and growth in the host plant. We sequenced the metagenome of E. solidaginis and wEsol and assembled and annotated the genome of wEsol. The wEsol genome has an assembled length of 1.66 Mbp and contains 1878 protein-coding genes. The wEsol genome is replete with proteins encoded by mobile genetic elements and shows evidence of seven different prophages. We also detected evidence of multiple small insertions of wEsol genes into the genome of the host insect. Our characterization of the genome of wEsol indicates that it is compromised in the synthesis of dimethylallyl pyrophosphate (DMAPP) and S-adenosyl L-methionine (SAM), which are precursors required for the synthesis of cytokinins and methylthiolated cytokinins. wEsol is also incapable of synthesizing tryptophan, and its genome contains no enzymes in any of the known pathways for the synthesis of indole-3-acetic acid (IAA) from tryptophan. wEsol must steal DMAPP and L-methionine from its host and therefore is unlikely to provide cytokinin and auxin to its insect host for use in gall induction. Furthermore, in spite of its large repertoire of predicted Type IV secreted effector proteins, these effectors are more likely to contribute to the acquisition of nutrients and the manipulation of the host's cellular environment to contribute to growth and reproduction of wEsol than to aid E. solidaginis in manipulating its host plant. Combined with earlier work that shows that wEsol is absent from the salivary glands of E. solidaginis, our results suggest that wEsol does not contribute to gall induction by its host.

The parent, child and young person experience of difficult venous access and recommendations for clinical practice: A qualitative descriptive study.

Explore the parent and child/young person experience of difficult venous access and identify ideas and preferences for changes to clinical practice. Peripheral intravenous catheter insertion is one of the most common invasive procedures in hospitalised paediatric patients. Multiple insertion attempts in paediatric patients are common and associated with pain and distress. Little research has explored the parent and child/young person experience of difficult venous access nor sought to identify their suggestions to improve clinical practice. Qualitative description. A purposive sampling approach was used to identify children and young people with experience of difficult venous access and their parents. Semi-structured interviews were conducted, with sample size based on data saturation. Transcripts were analysed using thematic analysis. There were 12 participants, seven parents and five children/young people (five parent/child dyads and two individual parents). Analysis of the data revealed three main themes: (1) Distress-before, during and after (2) Families navigating the system: the challenging journey from general clinician to specialist and (3) Difficult venous access impacts both treatment and life outside the hospital A pre-determined theme, (4) Recommendations for good clinical practice is also described. Multiple attempts to insert a peripheral intravenous catheter are a source of substantial distress for children/young people, leading to treatment avoidance. Effective interpersonal skills, providing choice and avoiding frightening language are important to minimise distress. Clinicians without specialist training should assess each child's venous access experience and consider immediate referral to a specialist if they have a history of difficult venous access. Cultural change is required so clinicians and healthcare services recognise that repeated cannulation may be a source of psychological distress for children/young people.

The Platysma Contraction Pattern in Healthy Adults: A Vector Analysis Study.

The platysma muscle's role in lower face dynamics is complex. Multiple insertion points to soft-tissue structures at various levels in the lower face create a multifaceted contraction pattern. To avoid adverse effects in cosmetic procedures when targeting the platysma, its anatomy and physiology must be understood. Clinical observations hint at a bidirectional contraction pattern. Eighteen healthy volunteers (13 women and five men) with a mean age of 44.2 ± 10.1 years were enrolled. Skin displacement vector analysis was used on maximal platysma contraction to characterize and calculate the movement of the neck and lower face skin. In all of the participants, a bidirectional movement of the skin was observed: the skin of the lower face and inferior to the jawline moved caudally, whereas the skin of the lower neck moved cephalad. Both movements converged at a line situated at 54% ± 10% and 55% ± 8% of the length between the clavicle and the inferior base of the ear lobe in men and women, respectively ( P = 0.70). The platysma is a bidirectional muscle with a line of convergence. Whereas the superior portion acts as lip depressor, the lower portion elevates the skin of the upper chest and lower neck. This transition can explain some of the clinically observed adverse effects of neuromodulation of the neck area. It can potentially direct neuromodulation injections to focus above the convergence line to better address lower face descent.

Predictors of Suboptimal Reperfusion and Short Term Mortality in Patients Presented With Acute ST-Segment Elevation Myocardial Infarction and Treated With Primary Percutaneous Coronary Intervention

Background: Predictors of Suboptimal reperfusion are still unclear. Aim: This study aimed to determine the factors that may indicate suboptimal reperfusion and short-term mortality in patients who were diagnosed with acute ST-segment elevation myocardial infarction (STEMI) and underwent primary percutaneous coronary intervention (pPCI). Patients and methods: This multicenter prospective comparative study, conducted at Benha University hospitals and National Heart Institute, included 400 patients (age<18 years and both sex) with acute STEMI, who were treated with PPCI. They were divided equally into 2 groups; suboptimal and optimal reperfusion groups (TIMI < III Vs.TIMI III respectively). Clinical data was collected. ECG, laboratory investigations, echocardiographic study, PPCI and 6 months follow up were done to all patients included. Results: This study showed that advanced age (60.4±8.2), family history of CAD, dyslipidemia, being diabetic, prolonged Pain to PCI time, higher random blood sugar (RBG) at the time of presentation, Killip class >1, heavy thrombus burden, prediltation, multiple stents insertion and longer stent length are predictors of the SOR after PPCI. While, Patients on long term beta blockers (BB) and angiotensin-converting enzyme inhibitor (ACEI) are less likely to develop SOR. Furthermore, patients with SOR are more likely to develop in-hospital arrhythmias, heart failure, acute mitral regurge and inhospital mortality. Additionally, it increases 6-months risk of reischemia and mortality. Conclusions: Predicting the occurrence of no-reflow following pPCI can be achieved by considering various factors, such as clinical data, laboratory results, angiographic features, and procedural characteristics. Heavy thrombus burden, prediltation, dyslipidemia, longer stent length, pain to PCI time and RBG >300 (mg/dL) at the time of presentation were found to be the most predictable variables to SOR. Long term use of BB and ACEI were found to be significant independent factors that decreased the likelihood of TIMI9h were considered the most predictable variables to mortality in no reflow.

Endoplasmic Reticulum Stress and Cellular Homeostasis in Genetically Engineered Porcine Donors for Xenotransplantation.

Genetically engineered pigs with multiple gene deletions and insertions are predicted to extend porcine to human xenograft survival. Several genes have been successfully knocked out and inserted, yet more have failed to produce viable animals for unexplained reasons. The effects of gene editing on cellular homeostasis may be the cause of reduced embryo fitness, failed pregnancies, or poor piglet viability. The elements of cellular dysfunction such as endoplasmic reticulum stress and oxidative stress induced by gene editing may additively affect the quality of genetically engineered cells to be used for cloning. Evaluating the impact of each gene edit on cellular fitness for cloning will allow researchers to maintain the cellular homeostasis of engineered cells that were validated as candidates for cloning and the production of porcine organ donors.

Defining Successful Intubation on the First Attempt Using Both Laryngoscope and Endotracheal Tube Insertions: A Secondary Analysis of Clinical Trial Data

Successful intubation on the first attempt has historically been defined as successful placement of an endotracheal tube (ETT) using a single laryngoscope insertion. More recent studies have defined successful placement of an ETT using a single laryngoscope insertion followed by a single ETT insertion. We sought to estimate the prevalence of first-attempt success using these 2 definitions and estimate their associations with the duration of intubation and serious complications. We performed a secondary analysis of data from 2 multicenter randomized trials of critically ill adults being intubated in the emergency department or ICU. We calculated the percent difference in successful intubations on the first attempt, median difference in the duration of intubation, and percent difference in the development of serious complications by definition. The study population included 1,863 patients. Successful intubation on the first attempt decreased by 4.9% (95% confidence interval 2.5% to 7.3%) when defined as 1 laryngoscope insertion followed by 1 ETT insertion (81.2%) compared with when defined as only 1 laryngoscope insertion (86.0%). When successful intubation with 1 laryngoscope and 1 ETT insertion was compared with 1 laryngoscope and multiple ETT insertions, the median duration of intubation decreased by 35.0 seconds (95% confidence interval 8.9 to 61.1 seconds). Defining successful intubation on the first attempt as placement of an ETT in the trachea using 1 laryngoscope and 1 ETT insertion identifies attempts with the shortest apneic time.

Exploring Australian emergency department clinicians' knowledge, attitudes and adherence to the national peripheral intravenous catheter clinical care standard: A cross-sectional national survey.

This study aimed to (i) capture clinicians' knowledge, attitude and adherence to the first Australian national peripheral intravenous catheter (PIVC) Clinical Care Standard, (ii) examine the instrument performance of the knowledge related questions and (iii) explore the educational needs for, and barriers to, Standard adherence among Australian ED clinicians. A cross-sectional national online survey was conducted from March to June 2022, using a snowball sampling method. The survey used 5-point Likert scales and multiple-choice questions to capture respondents' knowledge, attitude and adherence to the Standard as well as the educational needs for, and barriers to, Standard adherence. In total, 433 ED nurses and doctors responded. Although nearly half (n = 206, 47.6%; 95% confidence interval [CI] 55.5-65.8) of respondents claimed that they were unfamiliar with the Standard, questions on PIVC knowledge yielded that most respondents had adequate knowledge of most of the key standards. Respondents' attitudes towards multiple intravenous insertion attempts and ongoing PIVC competency monitoring are not in agreement with the Standard. Self-reported practices regarding routine insertion of idle catheters (55%; 95% CI 49.9-59.9), using antecubital fossa as the first insertion site (84%; 95% CI 80-87), insertion without confidence (46%; 95% CI 41.2-51.1) and lack of routine reviewing the ongoing needs of PIVC (40%; 95% CI 35.3-45.1) were not aligned with the Standard. Unawareness of the Standard and non-practical recommendations were rated as the top barriers to Standard adherence. The findings of the survey suggest that the Standard may need modification to align with the needs of ED clinicians. Future studies need to explore the applicability and relevancy of some recommendations in the ED settings as they may cause low adherence to the Standard.

Thermal and Flow Analysis of TiO2 Nanofluid Flow in Circular and Square Ducts with Multiple Twisted Tape Inserts

A numerical study is conducted to observe the thermal and flow performance of TiO2 nanofluid in the circular and square ducts with different twisted tape arrangements. The presence of the twisted tape in the tube induces swirl flow, which aids in the heat transfer, but at the penalty of a higher friction factor. The results also reveal that the maximum Nusselt number enhancement is obtained in the circular tube when the counter-triple twisted tape arrangement (C-TTs) is adopted while it is co-triple twisted tape arrangement (Co-TTs) for the case of square duct. Besides, the highest friction factor is observed for the Co-TTs for both circular and square ducts. The heat transfer and friction factor in the circular duct are greater than that of the square duct. The highest thermal performance factor of 1.286 is obtained when the single twisted tape and 1.5% nanofluid are used in the circular tube. However, multiple twisted tape inserts in the square duct contributes to improved thermal performance at a relatively lower friction factor when compared to the circular tube. Therefore, it is recommended to implement the square duct with multiple inserts for compact or microchannel heat exchanger for heat transfer application.

Clonal transmission of polymyxin B-resistant hypervirulent Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaKPC-2 in a tertiary hospital in China

BackgroundThe prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak.ResultsThe minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored blaKPC-2, blaNDM-1, and the virulence-related genes prmpA, prmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8–20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying blaKPC-2, blaNDM-1, blaSHV-12, blaLAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance.ConclusionsPolymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Reactions Involving Multiple Isocyanide Insertions

AbstractIsocyanides (isonitriles or carbylamine) have been intensively used in organic synthesis to prepare a diverse variety of N‐heterocycles on the basis of the carbene‐like reactivity of their divalent carbon atom. Isocyanides participate in a variety of reactions involving one, two, or more isocyanides. Compared to the popularity of single isocyanide reactions, few examples of reactions involving two or more isocyanides have been reported. In this review, we categorized and classified the literatures of reactions involving two or more isocyanides especially double isocyanide insertions under metal‐catalyzed or metal‐free conditions from 2014 to 2022.magnified image

Assembly-free quantification of vagrant DNA inserts.

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called "vagrantDNA" and code to simulate test data sets.

In Vivo Tracking of Persistent Organic Pollutants via a Coaxially Integrated and Implanted Photofuel Microsensor.

In vivo tracking of persistent organic pollutants (POPs) is of great significance for assessing their risks to the ecological environment and human health. However, existing in vivo POPs detection methods are limited by the lethal sampling of living organisms, complex sample preparation processes, or bulky testing equipment. Photoelectrochemical (PEC) sensing with the merits of high sensitivity and simple equipment is a fast-developed method for in vivo analysis. A major obstacle for in vivo PEC sensors is the separated implantation of multiple electrodes and a light source, which raises concerns like multielectrode biofouling and electroactive molecules interference in the complex environment, uncertain electrode implant distance, and multiple insertion operations. Here, a coaxially implanted photofuel microsensor was developed by hiding the optical fiber-based photoanode inside the glass capillary-based biocathode, and the model target PCB77 can be detected with an ultralow detection limit (2.8 fg/mL). This unique photoanode-biocathode-light source integrated structure ensures excellent selectivity, good antifouling ability and biocompatibility, high accuracy, and less implant mechanical damage. Combined with a handheld pH meter, our sensor achieved convenient and direct tracking of the bioaccumulation levels of PCB77 in freely swimming fish.

Diversity of viroids infecting grapevines in the South African Vitis germplasm collection.

Seven viroid species and one putative viroid species have been reported to infect grapevine namely, hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Japanese grapevine viroid (JGVd), grapevine latent viroid (GLVd), and citrus exocortis viroid (CEVd), as well as a grapevine hammerhead viroid-like RNA (GHVd), so far. In this study, RNA sequence (RNA-Seq) data, from 229 Vitis accessions from the field-maintained vineyard of the South African Vitis germplasm collection, were analysed to determine the diversity of the viroids present. Five of the seven known grapevine-infecting viroids and one putative grapevine-infecting viroid species were very commonly found, with 214 of the 229 samples containing at least one viroid species. HSVd, GYSVd-1, GYSVd-2, AGVd, and JGVd, as well as GHVd, were identified in the RNA-Seq data of the samples and confirmed using RT-PCR and Sanger sequencing. The HSVd sequences indicated the presence of two variants, with one showing multiple nucleotide insertions. AGVd and GYSVd-2 did not display significant sequence diversity, confirming past international studies. GYSVd-1 occurs as four major variants worldwide and representatives of all four variants were identified in this vineyard. This is the first report on the diversity of viroids infecting grapevine in South Africa and the first report of JGVd outside of Japan and GHVd in South Africa. Further studies are needed to fully assess the population and to identify potentially new viroid species.

Impact of Multiple Insertions and Withdrawals, and Vibration on Passive Intermodulation in Coaxial Connectors

Passive intermodulation (PIM) interference has a significant impact on the transmission quality of radio frequency (RF) communication systems. Coaxial connectors are one of the main nonlinear sources that cause PIM interference in communication systems. In addition, the degradation of connectors caused by multiple insertions and withdrawals, and vibration tends to degrade the PIM performance. In this current work, the mechanism of the influence of these factors on PIM was analyzed. A polynomial model was developed based on the tunneling effect in order to describe the PIM performance of coaxial connectors with various levels of degradation caused by multiple insertions and withdrawals, and vibration. A series of two-tone PIM tests on pristine connectors and degraded connectors were conducted. The test results show good agreement with the model results. This work serves to provide a theoretical basis for evaluating the impact of these factors on the PIM performance of coaxial connectors.

Site-specific phosphorylation of VDAC1 decreases its susceptibility to oligomerization.

The voltage-dependent anion channel 1 (VDAC1) is the primary pathway for metabolite and ion flux across the outer mitochondrial membrane. Oligomerization of VDAC1 that results in the formation of a mega-pore is a critical step in the release of cytochrome c and mitochondrial DNA that triggers apoptosis and inflammasome activation, respectively. However, the underlying mechanism that leads to mega-pore formation is unresolved. Previously, we identified phosphorylation of VDAC1 at S35 to be associated with cardioprotection. Here we report a novel finding that S35 phosphorylation regulates VDAC1 oligomerization and, subsequently, mega-pore formation. In 90 min recordings of channel activity of recombinant VDAC1 in planar lipid bilayers, wild-type (WT) VDAC1 exhibited cumulative current increases due to multiple channel insertions. In 50% of the recordings, a dramatic increase in cumulative channel conductance was observed (>1.8 µS). This was accompanied by a progressive shift at baseline, suggestive of high local ionic accumulation at the bilayer surface. These distinct biophysical characteristics were attributed to mega-pore formation. The probability of similar cumulative large conductances with associated baseline shifts was reduced in the phosphomimetic S35E VDAC1 (20% vs 50% in WT), while it was increased in a non-phosphorylatable mutant S35A (90% vs 50% in WT). In VDAC1 knock-out H9c2 cells expressing WT or mutant (S35A/S35E) VDAC1, 400 µM H2O2 induced the formation of VDAC1 oligomers. However, compared to WT, the formation of trimers and higher order oligomers was significantly decreased in cells expressing the S35E mutant, while it was significantly increased in cells expressing the S35A mutant. These effects are consistent with the corresponding biophysical characteristics observed in the bilayer experiments. Our results suggest that phosphorylation of S35 decreases, while dephosphorylation increases, the susceptibility of VDAC1 to oligomerization and mega-pore formation.

Membrane mechanics regulate the cooperativity of proteins in curvature sensing.

Recruitment of proteins to a membrane surface is critical for many biological processes, such as clathrin-mediated endocytosis and signaling. When interacting with the lipid membrane, many proteins exhibit different affinities to membranes with different curvature, known as curvature sensing. They also generate membrane curvature (curvature induction) by a variety of mechanisms, including the insertion of amphipathic helices. Here we use a continuum membrane model to study how helix insertions can alter the membrane energetics and how the mechanical feedback of the membrane regulates the helix insertions. Our membrane model has two coupled layers of triangular mesh to represent the two monolayers of the bilayer membrane, capturing membrane thickness and tilt. We analyzed the membrane deformation around one helix insertion and compared it to molecular dynamic simulations to optimally define the lipid tilt energies. We then investigate the interactions between insertions and membranes by considering the copy number of insertions and their interval distance, membrane tension or stress before the insertions, membrane curvatures, and lipid composition. Our results showed that multiple insertions exhibit a mechanism of cooperativity; each binding event is not independent of previous events. Our results show that this cooperativity is dependent on membrane tension and curvature. In comparison to experimental data, we validated our simulation results and analyzed the role of lipid flip-flop across the bilayers. Quantifying how protein and membrane interactions feedback on one another is critical for understanding the dynamics and control of proteins that remodel membranes through localization and self-assembly in various cellular processes.

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

BackgroundDespite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming.ResultsIn this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes.ConclusionsThe novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

Molecular characterisation of a meropenem-resistant Haemophilus influenzae

Background: Haemophilus influenzae β-lactam resistance is mediated by production of β-lactamase and mutations in penicillin-binding protein 3 (PBP3, encoded by ftsI). Only one of the 646 patient-unique H. influenzae isolated in our laboratory between 2012–July 2022 was meropenem (amoxicillin, co-amoxiclav and ceftriaxone) resistant. Methods: Identification and susceptibility of this meropenem-resistant H. influenzae (MRHI) was confirmed by disk diffusion and E-test (bioMérieux) (CLSI M100). WGS was performed on GridION (Nanopore) and reference mapped with Geneious Prime 2022.2. PCR and Sanger sequencing was used to confirm results of WGS. Results: The MRHI isolate was β-lactamase negative and non-typeable. ftsI subgroup IIc1 mutations were present, signifying previously described low level PBP3 alterations that should not solely account for carbapenem resistance. Group VII2 acrR (repressor of AcrAB efflux pump) substitutions were observed but without the reported carbapenem resistance associated frameshift mutation. There were also multiple substitutions and insertions in ompP2 (outer membrane protein P2) porin, associated with carbapenem resistance.2 The 100% concordance between Sanger sequencing and Nanopore increases our confidence for application of this technology for future investigations. Conclusion: Meropenem resistance in this isolate is likely multifactorial, with PBP3 alterations and decreased influx as contributing factors.