C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.Gene editing in  C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.Germlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode  PLOS Genetics Methods paper.This is a CRISPR has revolutionized genetics by providing a simple method for targeted mutation, deletion or tagging of genes . Since iC. elegans, the Cas9 endonuclease is introduced into the germline via microinjection of either protein or DNA. In the protein method, Cas9 protein complexed in vitro with guide RNA is directly injected into the gonad along with a DNA repair template .,18.mex-5.,18.mex-dpy-10 sgRNA. Animals were injected with a plasmid expressing a dpy-10 single-guide RNA (sgRNA). We monitored array-positive progeny for dumpy (Dpy) or rolling (Rol) phenotypes consistent with mutation of dpy-10. In the strain bearing the germline-optimized Cas9 construct (EG9615), ~25% of array-bearing F1 animals (18/69) exhibited a Dpy or Rol phenotype. To determine if these were heritable mutations, we singled F1 animals. Roughly half of these (8/15 singled F1s) were caused by germline mutations, that is, the singled animals produced either 100% Dpy or one quarter Dpy progeny, consistent with germline mutation. By contrast, in strains expressing the Cas9 cDNA construct, we failed to detect dpy-10 mutant animals among similar numbers of array positive progeny; this strain was not characterized further. Together, these data indicate that a single-copy transgene can express sufficient Cas9 to introduce cuts in the genome, but it must be licensed for germline expression.Each Cas9 expression strain was tested for basal activity by testing for double-strand break formation using the high-efficiency unc-119(+) transgene nested in an intron of GFP  or 1xGreen co-injection markers [2 ng/μl pCFJ91(Pmyo-2::gfp) + 8 ng/μl pCFJ421 (Peft-3::gfp::h2b)]. For CRISPR injections with a single plasmid containing both the repair template and guide RNA expression cassette, the plasmid was included at 65 ng/μl. For CRISPR injections with the repair template and guide RNA expression cassette on different plasmids, the repair template was included at 60 ng/μl and the guide RNA expression plasmid was included at 30 ng/μl. When included, Cas9 expression plasmids were included at 30 ng/μl (cDNA) or 2 ng/μl (with smu-2 introns); higher levels of the germline-licensed Cas9 transgene were toxic. If needed to bring injection mix concentrations to 100 ng/μl, pBluescript(sk+) was used as “stuffer” DNA. After injection, P0s were either singled to fresh OP50 plates (when quantifying event frequency per injected P0) or pooled in groups of 3–5 (for construction operations) and incubated at 25°C for 3–10 days prior to screening.All injections were conducted into young adult (<24 hours) hermaphrodites reared at 15°C on either OP50 (wild type worm strains) or HB101 (eft-3::Mosase), 10 ng/μl 1xRed markers, and 10 ng/μl pMA122 (Phsp-16.41::peel-1) . MiniMos injections were conducted with young adult EG9814 [unc-119(ox819) III] worms. MiniMos injection mixes contained 50 ng/μl pCFJ601 (Peft-3::Mosase), 40 ng/μl pMLS714 (miniMos targeting vector), and 10 ng/μl 1xRed markers. Initial MosSci inserts (oxSi1196 and oxSi1091) were built using the standard pCFJ150 targeting vector. To allow removal of Cbr-unc-119 after transgene insertion, we built general targeting vectors for MosSci at the ttTi5605 locus (pMLS640) and for MiniMos (pMLS713) containing a lox2272-flanked Cbr-unc-119 cassette. In all cases, genomic inserts were identified by screening for Unc-119+ animals that lacked extrachromosomal array markers. The floxed Cbr-unc-119 was removed from strains by injecting an array containing 50 ng/μl pDD104 (Peft-3::Cre) and selecting for unc F2 progeny.All MosSci injections were conducted with young adult EG6249 worms. MosSci injection mixes contained 25–40 ng/μl ttTi5605-targeting vector, 50 ng/μl pCFJ601 , 10 ng/μl either pMLS716 (Phsp-16.41::Cre + lox2272 flanked Cbr-unc-119) or 2 ng/μl pMLS791 (Phsp-16.41::Cre + Pmyo-2::2x-nls-cyOFP + lox2272 flanked Cbr-unc-119) + 10 ng/μl 1xRed marker. Cas9 activity was provided by the integrated Cas9 transgene. Injected P0 were incubated at 25°C for 10 days. Inserts were selected by unc-119 rescue and the absence of fluorescence array markers. To remove the Cbr-unc-119 marker, larval animals were heat shocked at 32°C for 2 hours. unc-119- animals were selected by phenotype from the F1 progeny of heat-shocked animals.Cas9+, dpy-10 sgRNA plasmid to improve our dynamic range: 10 ng/μl pMLS597 (PU6::sgRNA(dpy-10)), 10 ng/μl 1xRed markers, and 80 ng/μl stuffer DNA. For screening miniMos-based Cas9 strains, worms were injected with 90 ng/μl pMLS597 (PU6::sgRNA(dpy-10)) and 10 ng/μl 1xRed markers. The higher concentration of pMLS597 resulted in a higher rate of germline mutagenesis of dpy-10 among array(+) animals . Injected P0 animals were pooled on fresh OP50 plates and incubated at 25°C for 2–3 days. Array(+) F1 animals were selected by fluorescence from the co-injection markers. Selected worms were later scored for Dpy and Rol phenotypes under white light.For initially screening MosSci Cas9 strains, worms were injected with a low concentration of 2 generation.For quantifying insertion frequency at multiple target loci in EG9747, worms were injected with 65 ng/μl targeting vector, 10 ng/μl 1xRed markers, and 25 ng/μl suffer DNA. For quantifying insertion frequency in miniMos insertion strains, worms were injected with 90 ng/μl targeting vector (pMLS338) and 10 ng/μl 1xRed markers. We used only 65 ng/μl targeting vector when quantifying insertion frequency in EG9747 so that the targeting vector concentration in these injections matched the targeting vector concentration used for quantifying insertion frequency at these sites using plasmid-borne Cas9 . After injection, P0 animals were singled to OP50 plates and incubated at either 25°C or room temperature (20°C) until starvation. Starved plates were inspected for array+ and GFP+ animals. Insertion frequency was calculated as (number of plates with GFP + worms) / (number of successful injections), where a ‘successfully injection’ is an injection resulting in array+ worms in the FW03F9.11 on chromosome V was silenced by insertion of the fluorescent marker Pmyo-2::2xNLS-cyOFP::let-858 3’UTR. To genetically mark the Cas9 transgene we inserted the cyOFP marker at two sites linked to W03F9.11. Both insertions resulted in silencing of Cas9 activity. The first landing site was 13.7 kb away between W03F9.4 and ttb-1 (bp154310). The second landing site was 141 kb away between nra-4 and flp-34 (bp1037379).Cas9 activity from the miniMos insertion at unc-32::GFP(Cbr-unc-119+) alleles created in either the EG9886, EG9875, or EG9880 background. Cohorts of ~20 homozygous young larvae (L1 –L2) of each strain were either incubated at room temperature or at 32°C for 2 hours (“heat shock”). After adulthood, eggs were collected from each cohort, and each hatched animal was scored for the presence of Unc-119(-) phenotype.For quantifying Cre activity, we used S1 TableNumber of injected P0 worms (EG9747) that yielded at least one GFP insertion event on primary or secondary plates. ‘Primary + Secondary’ tallies P0s for which GFP+ worms were found on both the primary plate founded by the single injected P0 and on the secondary plate founded by 20 array+ GFP- progeny of the originally injected P0 (~F3). ‘Primary Only’ tallies P0s for which GFP+ worms were found on the primary plate but not on the secondary plate. ‘Secondary Only’ tallies P0s for which GFP+ worms were not found on the primary plate but were found on the secondary plate. ‘Neither’ tallies P0s for which GFP+ worms were not found among the progeny on either the primary of secondary plate.(PDF)Click here for additional data file.S2 Tabledpy-10’: P0s injected with a dpy-10 sgRNA expressing plasmid, ‘+’ indicates the presence, and ‘-‘ indicates the absence of Dpy and Rol worms among the progeny of those P0s. ‘GFP inserts’: number of lines yielding unc-32::GFP insertions over total injected lines.Summary of analyses of 14 Cas9 miniMos integration strains. ‘Strain’ is the isolation name. ‘chrom’ and ‘gene’ identify the location of the insert determined by inverse PCR, ‘unmapped’ means the inverse PCR failed or was ambiguous. PCR confirmation: ‘+’ indicates position was confirmed by locus-specific PCR, ‘-‘ indicates confirmatory PCR failed. ‘n.t.’, not tested. ‘cuts (PDF)Click here for additional data file.S3 Tableunc-119 selections: EG9747 chromosome II, EG9881 chromosome III, EG9887 chromosome I, EG9891 chromosome V.Bolded strains are the most useful for CRISPR modifications using (PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Figunc-119(ed3) allele was generated using EMS, and is likely to have other mutations in its background. Because CRISPR edits should be generated in a wild-type background that has not been exposed to a chemical mutagen, we generated a new unc-119 mutation in a wild-type background. N2 worms were injected with a plasmid encoding Cas9 and a guide RNA targeting the same region of unc-119 as ed3. Sequencing of ox819 revealed an 11 base-pair deletion starting at V110 (UNC-119a) straddling the targeted cut site which leads to a frameshift and stop after 27aa.The classic (PDF)Click here for additional data file.S2 Figmyo-2::2xNLS-cyOFP::let-858 3’UTR transgene (EG9882). Fluorescence is brightest in L3 and L4 larvae and is dim in adults. (Bottom) cyOFP has a broad excitation range (maximum from ~480–520) and orange fluorescence emission (590 nm peak) that can be observed using a mercury lamp and standard GFP longpass filter sets on a dissecting scope or using 488 nm or 514 nm lasers with an orange-red bandpass or long-pass emission filter  on a confocal microscope. Emission and excitation spectra from fpbase.org.(Top) Fluorescence pattern of nuclear localized ‘cyan-excitable Orange Fluorescent Protein’ (cyOFP) in pharynx of an L4 larva from the P(PDF)Click here for additional data file.S3 Fig(A and B) Dead-end target sites: 20 EG9881 animals were injected with a guide RNA and repair template for tagging unc-32 with gfp. At starvation of the primary plates, array+, gfp- animals were singled to generate secondary plates, and the plates were incubated until starvation. Some lines failed to generate GFP+ animals (8/20 lines) by the time the secondary plate starved. These lines were analyzed by PCR amplification of the targeted insertion site (A) and then followed by sequencing (B). (A) PCR amplification of the unc-32 target locus. Expected amplicon sizes for the native locus and for a complete gfp + Cbr-unc-119 insert are indicated. (B) Sequencing results for the two unc-32 loci in each strain. (Top) the native unc-32 target site is shown with the guide RNA binding site and Cas9 cut site highlighted. For each strain, the sequences for both chromosomes are shown. Unique alleles are individually color coded to highlight sibling relationships. Strains 1, 2, 4, 6 and 7 likely originated from a single parent, and strains 3 and 8 likely originated from a single parent. These sequences document six different healing events: three chromosomes healed by NHEJ , one chromosome (green) incorporated a truncated GFP, one chromosome (brown) inserted 4 nucleotides at the cut site, and one chromosome (pink) inserted a novel segment with sequence variations (underlined). It is possible this sequence represents a de novo event segregating in this strain. (C) Location of two ‘dark inserts’. Two strains with off target insertions of unc-119(+) were located by whole genome sequencing (Illumina). After aligning all reads to the reference C. elegans genome, reads that failed to align to the genome were aligned to the sequences of all injected plasmid species. From this mapping, we determined which array sequences were present in the genome. To locate the junctions between the plasmid and the genome, we identified regions by inspection where the plasmid reads contained unmapped tails. BLAST analysis of the unmapped tails was used to determine the insertion site. The insertion site of one of the off-target events is in a central intron of the rict-1 gene. The sequences flanking the second off-target insertion are present at multiple loci in the genome, preventing exact mapping. (i). Schematic representation of the CRISPR targeting injection mix containing a targeting plasmid and multiple array marker plasmids. (ii). Schematic representation of the off-target insertion of the entire gfp::snb-1 targeting plasmid into the gene rict-1 on chromosome II. (iii). Schematic representation of the off-target insertion of a portion of the unc-32::gfp targeting plasmid into a non-unique genomic location. The insertions were not at sites obviously similar to the predicted guide RNA binding sites.(PDF)Click here for additional data file.S4 FigoxTi’). oxSi1106 is a single-copy insertion at the Mos1 insertion site ttTi5605. Also depicted are prominent MosSCI sites (light blue) and ‘universal MosSCI ‘sites (dark blue) [ttTi5605 arms for homologous recombination insertion and are usually flanked by NeoR and unc-18(+) transgenes (see www.wormbuilder.org website).Insertion sites of the integrated Cas9 alleles (red) in the genome. The locus into which the miniMos-Cas9 transposon inserted is written above (for example ‘W01A8.6’), and the original, unmodified, transposon insertions are named (‘rk blue) ,27. ‘Uni(PDF)Click here for additional data file.S5 FigInsertion sites are ordered by chromosome number (‘Chr’) and site into which the miniMos inserted (red). Strain and name of modified miniMos element are provided below (black). The chromosome II site is a MosSci insertion into the ttTi5605 site and does not have a Cre or orange fluorescent protein (‘cyOFP’) transgene.(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.


Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato.RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2 RNA silencing refers to one of the cellular pathways that regulates gene expression through specific mechanisms acting on RNA and is based on the sequence specificity of nucleic acids. RNA silencing is considered to have evolved to regulate gene expression both in the nucleus and the cytoplasm of almost all eukaryotes. Among many functions, RNA silencing serves as an antiviral mechanism in plants. The replicative forms of ssRNA viruses or other sources of dsRNA such as regions with strong secondary structures result in double-stranded RNA that triggers RNA silencing. This viral dsRNA is recognized and processed into viral siRNAs (vsRNAs) by the Dicer-like (DCL) enzymes. vsRNAs are then associated with argonaute (AGO) proteins, which are the central molecule of the RNA induced silencing complex (RISC) and guide the RISC to the viral nucleic acids for target cleavage or translational inhibition .Plant viruses evolved viral suppressors of RNA silencing (VSR) proteins to counteract RNA silencing. VSRs are very diverse proteins and inhibit different steps of RNA silencing such as the generation of vsRNAs, the sequestering of vsRNAs, the formation of RISC complexes or the inhibition of a pre-assembled RISC ,2.Arabidopsis thaliana encodes 10 AGO proteins that are shown to be involved in different RNA silencing pathways and AGO1 was first shown to be required for antiviral defense -UTP-labeled single stranded in vitro transcribed RNA probes corresponding with the negative strand of the P1 coding region of the SPMMV genome.RNA was isolated using the 2 × PK buffer  supplemented with 10 μg/mL proteinase K. The reactions were incubated at 55 °C for 10 min then extracted with phenol-chloroform and nucleic acids were precipitated with 2 volumes of EtOH. The RNA was separated by 2.2 M formaldehyde and 1.2% RNA was isolated with a 2 × PK buffer  supplemented with 10 μg/mL proteinase K from leaves or the eluates of immunoprecipitations and then separated on a 10% acrylamide and 8 M urea containing denaturing gel and blotted to an Amersham Hybond-N membrane. The membranes were hybridized with [γ-32P]-ATP-labeled LNA or DNA oligonucleotides. Proteins were isolated using the 4 M urea, 50 mM Tris-HCl, 100 mM NaCl and 20 mM EDTA pH8.0 containing buffer. A total of 20 μg protein was loaded to a 6% or 10% acrylamide SDS-PAGE gel then transferred to an Immobilon-P membrane (Millipore). The membranes were incubated with AGO1 (Agrisera) or AGO2 antibodies (19) in a TBS-TT buffer .2, 5 mM DDT and 0.5% Tween 20) and were incubated with AGO1 (Agrisera) or AGO2 antibodies [Extracts were prepared in an IP buffer  from mock and infected systemic leaves at 10 dpi. qRT-PCR experiments were performed as described by .


Temporal artery biopsy (TAB) is often performed by ophthalmology trainees without direct supervision. The traditional model of ‘see one, do one, teach one’ still prevails in most units. Whilst it is generally a safe procedure, damage to the temporal branch of the facial nerve has been reported when harvesting the frontal branch of the superficial temporal artery.A survey of trainees from Wessex, Wales, London and Severn deaneries was performed to look at current training techniques, anatomical knowledge and practice.38 trainees responded to the survey, with complete responses from 28 participants. Formal teaching of the anatomical considerations in TAB was not reported by any trainee, with informal teaching being standard practice. Whilst 61% of respondents reported having learnt about the anatomical ‘danger zone’ for facial nerve damage, 97% of trainees chose an incision that fell within this zone when given a choice between potential incision sites.TAB remains a largely trainee-taught, trainee-performed procedure. Most trainees are not aware of how to avoid the risk of damage to the temporal branch of the facial nerve. We suggest harvesting the parietal branch of the temporal artery via an incision outside the anatomical ‘danger zone’. In our experience, this is an easily taught technique that minimises the potential risk of damage to the frontal branch of the facial nerve. Despite recent advances in investigative options for the diagnosis of giant cell arteritis (GCA), temporal artery biopsy (TAB) remains a vital diagnostic tool . EffortsThe superficial temporal artery (STA) lies within the superficial temporal fascia (STF) and the temporal branch of the facial nerve (TFN) courses deeper within the fascia’s fibrofatty layer . The TFNThe anatomical ‘danger zone’ has been described as an area where the TFN and the FSTA are separated solely by the superficial temporal fascia . Yoon etOther authors have previously described a technique for harvesting the parietal branch, rather than the frontal branch, of the temporal artery . This caIn this study we investigated current training techniques, anatomical knowledge and practice in TAB to establish current practice.A survey was electronically distributed, using the Survey Monkey platform, to ~100 trainees from the Wessex, Wales, London and Severn deaneries during 2019.Questions asked included year of training, number of TABs performed, and the method of training received. Trainees were also required to select where they would make their initial incision based on a series of diagrams and were asked about their awareness of the anatomical ‘danger zone’. All questions were designed to allow the respondents to select from options.Our study adhered to the tenets of the Declaration of Helsinki. Consent was obtained from all individual participants included in the study.38 responses were received out of approximately 100 distributed surveys. Ten respondents started the survey but only completed the first few questions and were therefore excluded from the analysis, leaving 28 complete responses. Responses were received from trainees from ST2 to ST7 level with most being from ST7 trainees.The question concerning the type of training received revealed that 28 (100%) respondents received informal training from their colleagues in the format of ‘See one, do one, teach one’.8/28 trainees (28.6%) had performed between 1 and 5 temporal artery biopsies; 8/28 (28.6%) had performed between 5 and 10 temporal artery biopsies; 5/28 (17.9%) had performed between 10 and 20 temporal artery biopsies; 6/28 (21.4%) had performed greater than 20 temporal artery biopsies, whilst 1/28 3.6%) trainee had not yet performed a temporal artery biopsy as part of their training  reported having been taught about the concept of a ‘danger zone’ for injury to the temporal branch of the facial nerve (TFN) Fig. . 5/28 1 whereas This is the first multi-deanery study to investigate temporal artery biopsy training for ophthalmology trainees in the UK. Ophthalmologists in the UK are required to undertake a 7-year Ophthalmic specialist training programme which has an extensive curriculum comprising of a number of core learning outcomes that must be achieved by the end of the final year . A key dAt present, our results confirm that the most common approach to TAB training is in the format of ‘See one, do one, teach one’. This means that trainees are not receiving formal teaching. In other areas of ophthalmic surgical training such as cataract surgery, implementation of a structured surgical curriculum involving the use of wet labs and simulator training, has been demonstrated to reduce surgical complications . TraineeThe use of informal teaching is likely multifactorial and may result from the shared performance of the procedure across multiple specialities. A recent retrospective cohort study reviewing the specialities performing TAB over a 10-year period in Canada found that general surgeons performed the most temporal artery biopsies which was closely followed by ophthalmologists and plastic surgeons . It is nFrom the perspective of future practice, the use of informal teaching can lead to surgical procedures being influenced by the teaching surgeons’ personal preferences. This can result in less room to propose safer alternatives to a surgical technique. A safer approach to TAB would be to harvest the parietal branch of the superficial temporal artery, effectively eliminating the risk of damage to the facial nerve. This has previously been discussed by other authors , yet thiWhile many trainees are performing temporal artery biopsies before their final year, with some performing greater than twenty biopsies, the majority of respondents indicated that they would make their initial incision within the ‘danger zone’. This would suggest that the teaching received by trainees lacks emphasis on the ‘danger zone’ as an anatomical region that should be avoided where possible. Therefore, we propose mapping the ‘danger zone’ during preoperative planning of TAB and avoiding the ‘danger zone’ when performing temporal artery biopsy.Our survey reveals that the majority of trainees are aware of the anatomical ‘danger zone’, however less than one-fifth of respondents are aware of Pitanguy’s line. The concept of Pitanguy’s line does have its limitations, as the most at-risk temporal branch of the facial nerve typically has multiple rami crossing the zygomatic arch , 24. HowThere are some limitations to this study. A primary limitation is the generalisation of these results. This is because the survey was distributed to a limited number of deaneries and in addition not all trainees responded. Furthermore, we received a number of incomplete surveys which were excluded from the analysis. Nonetheless, our study provides an insight into how trainees are being taught to perform a TAB and assists in evoking discussion regarding how training can be made safer.Despite the development of formal surgical teaching and simulation in other branches of ophthalmic surgery, the teaching of TAB continues to be taught to trainees, by trainees via the traditional apprenticeship method. Harm to patients may be avoided by raising awareness of the ‘danger zone’ and harvesting the parietal branch of the superficial temporal artery. Formal teaching on how to map the ‘danger zone’ and Pitanguy’s line and a demonstration of the above technique would be a beneficial addition to teaching trainees how to perform a TAB. This is particularly relevant as we are currently seeing an expansion in different methods of training, including the use of online resources, as a result of the COVID-19 pandemic. This has allowed us to be more creative and resourceful in the way we approach teaching which is proving to be beneficial in many sectors. TAB is a suitable technique that can be optimised with the addition of virtual training, and this will hopefully make the procedure safer for patients as well as instil confidence in our trainees.Temporal artery biopsy (TAB) is generally a safe procedure to perform however, there is a risk of damaging the temporal branch of the facial nerve. There is currently no formal teaching of the procedure to ophthalmology trainees. The traditional model of ‘see one, do one, teach one’ still prevails in most units.This study highlights that TAB still remains a primarily trainee-taught, trainee-performed procedure. Ophthalmology trainees will benefit from formal teaching on the anatomical danger zone and how best to avoid it.


Cell cycle is regulated through numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest, or undergo apoptosis. Abnormal cell cycle regulation has been linked to many diseases. Thus, there is an urgent need to understand the diverse molecular mechanisms of how the cell cycle is controlled. RNA helicases constitute a large family of proteins with functions in all aspects of RNA metabolism, including unwinding or annealing of RNA molecules to regulate pre-mRNA, rRNA and miRNA processing, clamping protein complexes on RNA, or remodeling ribonucleoprotein complexes, to regulate gene expression. RNA helicases also regulate the activity of specific proteins through direct interaction. Abnormal expression of RNA helicases has been associated with different diseases, including cancer, neurological disorders, aging, and autosomal dominant polycystic kidney disease (ADPKD) via regulation of a diverse range of cellular processes such as cell proliferation, cell cycle arrest, and apoptosis. Recent studies showed that RNA helicases participate in the regulation of the cell cycle progression at each cell cycle phase, including G1-S transition, S phase, G2-M transition, mitosis, and cytokinesis. In this review, we discuss the essential roles and mechanisms of RNA helicases in the regulation of the cell cycle at different phases. For that, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. We also discuss the different targeting strategies against RNA helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on specific RNA helicases, and the therapeutic potential of these compounds in the treatment of various disorders. The cell cycle is a series of events that occur in the interphase and mitotic phase (M-phase) ,2. The i2 checkpoint bars the entry of the cell cycle into the mitotic phase if certain conditions are not met, which ensures that all of the genetic material has been replicated and is not damaged. A damaged DNA will halt the cell cycle to let the damaged DNA be repaired. The M checkpoint, also named as the spindle checkpoint, is a point close to the end of the metaphase stage to make sure whether each pair of sister chromatids is correctly anchored to the two spindle microtubules—if not, the cycle will be halted. Recent studies suggest that RNA helicases are involved in these processes.During the cell cycle, there are three checkpoints that occur at the end of G1 and the transition of G2/M, and in metaphase. The G1 checkpoint blocks the entry of the cell cycle into the S phase if a cell does not fulfill all the conditions, which lets the cell try to solve the conditions, or lets the cell enter into the G0 phase. The GN- and/or C-terminal extensions of most of the RNA helicases are able to interact with specific RNA or protein cofactors. This family of DEAD-box RNA helicases is able to unwind and restructure RNA molecules in an ATPase-dependent manner  complexes that are associated with RNA transcription, degradation, translation initiation, mRNA splicing, and ribosome biogenesis ,8,9. RNAt manner  13,14].,14.N- anRNA helicases play important roles in diverse biological processes. First, RNA helicases can regulate gene expression through catalyzing RNP complex rearrangements at the initiation of gene transcription and then are involved in gene post-transcriptional expression via regulation of ribosome biogenesis, RNA export, translation initiation and termination, and mRNA degradation . Most ofPkd1 mutant renal epithelial cells and tissues to regulate cystic renal epithelial cell proliferation and renal fibrosis through activation of the PKD-associated pathways, including ERK, mTOR, Rb, and TGF-β1 , pr, pr84], In the cell cycle, the G2 phase is the second gap for cells to prepare for mitosis (M phase). During the G2 phase, cyclin A is degraded, but cyclin B is synthesized, resulting in the association of Cdc2 with cyclin B, which is required for the initiation of mitosis. Cyclin B can be phosphorylated at Tyr15 by WEE1, a kinase of the WEE family, resulting in altering equilibria and affecting G2/M transition ,89. The DDX56 regulates diverse RNA metabolism, including transcription, translation, ribosome biogenesis, pre-mRNA splicing, and mRNA degradation. DDX56 is a component of free nucleoplasm 65S preribosomal particles, which may participate in the assembly of the 60S ribosomal subunit. DDX56 has also been reported to regulate ribosome assembly through its association with the Oct4 and Sox2 complex, which is necessary to maintain embryonic stem cells (ESCs) proliferation . DDX56 iFurthermore, some RNA helicases that we mentioned above are also involved in G2/M phase transition. It has been reported that knockdown of eIF4A3 decreases cell cycle arrest in the G2/M phase, but increases apoptosis . DHX33 aCytokinesis is the last step of the cell cycle in which the cell must devotedly split the chromosomes and cytoplasm to produce two daughter cells with equal contents . RNA helUAP56 and its paralog URH49 are 90% identical to each other. These RNA helicases are nuclear RNA helicases essential for mRNA export and splicing and piRNA biogenesis. They also localize at cytoplasm . KnockdoCaenorhabditis elegans . DD. DD34]. DDX3 has also been identified as a positive regulator of p21 ,42. OverThe important roles of RNA helicases in the regulation of the cell cycle, cell proliferation, cellular transformation, apoptosis, and cell adhesion and motility make RNA helicases an active targets of drug development for the treatment of viral infections, neurodegenerative diseases, and cancers. Based on the crystal structures and the mechanism(s) of activity of the enzymes, specific and selective inhibitors for RNA helicases have been designed. In general, inhibitors targeting RNA helicase activity could act via one or more of the mechanisms: (1) inhibition of the ATPase activity of RNA helicases by interference with ATP binding and result in limiting the energy necessary for the unwinding and translocation; (2) inhibition of the helicase activity by competitively occupying the RNA binding site of the RNA helicases; and (3) stabilization of RNA helicases onto RNA, resulting in the inhibition of translation initiation.RX-5902 is a first-in-class anticancer compound targeting phosphorylated p68 . RX-590218 is an eIF4A inhibitor, which has been identified from a high throughput screening (HTS) campaign. Compound 18 selectively acts against eIF4A through competitive inhibition of its ATPase activity , a small molecule, is a ring-expanded nucleoside (REN) analogue. RK-33 is designed to selectively inhibit DDX3 activity but no other DEAD-box RNA helicases by binding to the ATP pocket of DDX3  117]. T. T117]. Hippuristanol, as a polyoxygenated steroid, selectively inhibits eIF4A through binding to its C-terminal domain . HippuriBoth Silvestrol and CR-1-31-B have been identified as specific inhibitors of the eIF4A, which act as the part of the translation initiation complex that binds to the 5′-cap structure  32,73].,73.32,73Pateamine A (Pat A) is designed as a biologically active metabolite, which selectively stimulates the RNA binding activity of eIF4A, resulting in disruption of the eIF4F complex and leading to inhibition of cap-dependent translation. Pat A also increases the ATPase and helicase activities of eIF4A . Pat A cAs described above, different inhibitors of RNA helicases have been developed, which may potentially be used in disease treatment ,123,124.The DDX3 inhibitor, NZ51, is able to block the ATP-dependent helicase activity of DDX3. NZ51 suppresses DDX3 activity at low micromole concentration and displays anti-proliferative activity by inhibiting cell replication at the G1 phase in aggressive breast cancer cells ,49. HoweHippuristanol, as a pan eIF4A inhibitor, has been reported to inhibit human T lymphotrophic virus type 1-infected T-cell line and adult T-cell leukemia cell proliferation by promoting cell cycle arrest at the G1 phase and suppressing the expression of cell cycle proteins and cyclin-dependent kinases, and inducing apoptosis by decreasing the expression levels of Bcl-x, baculoviral IAP repeat, containing 3 X-linked inhibitors of apoptosis (xIAP) and caspase 8 and FADD like apoptosis regulator ,128. TreDDX5 (p68) can be phosphorylated at the Y593 site by growth factors, including platelet-derived growth factor, to increase cellular proliferation, epithelial to mesenchymal transition (EMT), malignant transformation, cell migration, and oncogenesis through translocation of β-catenin to nuclear and activation of cyclin D1, c-JUN, and c-MYC . RX-5902Caenorhabditis elegans [Recently, several studies have demonstrated that the DEAD-box RNA helicases involved in regulation of liquid–liquid phase separation (LLPS) play a crucial role in the formation of RNA-containing membrane-less organelles, including pericentriolar material (PCM), stress granules (SGs), P bodies, and P granules in  elegans .Pericentriolar material is defined as a matrix of proteins surrounding the two barrel-shaped centrioles during mitosis, which serves as a platform for protein complexes to regulate organelle trafficking, protein degradation, and spindle assembly. PCM are highly dynamic, spherical organelles without a membrane. Zwicker et al.  used theStress granules are RNPs that assemble in response to environmental stresses such as oxidative stress, heat shock, or osmotic shock. One component of yeast stress granules is Ded1p, also named DDX3 in mammalian. Temperature-driven liquid phase condensation of Ded1p induces the sequestration of housekeeping mRNAs and promotes an expanded heat shock response in program results of the preferential expression of stress proteins at 39 °C . TargetiP bodies are the large cytoplasmic granules, which are membrane-less cytoplasmic organelles that form via phase-separation once RNAs and nearby RBPs assemble into ribonuclear particle (RNP) granules. P bodies have all the key characters of LLPS, which are liquid-like, spherical, and dynamic, and can be dissolved by the alcohol 1,6-hexanediol. One of the key regulators that regulates P bodies’ assembly is the DEAD-box ATPase Dhh1 (DDX6 in humans). Dhh1, as an enhancer of decapping, is involved in the translational repression of mRNAs. Dhh1 is involved in phase separation and controls P bodies dynamics in vivo, which is mainly dependent on its RNA-stimulated ATPase activity. The ATPase activator Not1 dissolves Dhh1 droplets in vitro and inhibits P-body formation in vivo . P bodieCaenorhabditis elegans. P granules are membrane-less organelles that may assemble by intracellular phase separation. GLH-1, a germline putative RNA helicase, regulates the formation and disassembly of P granules coupling with distinct steps of its ATPase hydrolysis cycle [P granules are RNA/protein condensates in the germline of is cycle . PGL-1 ais cycle . In addiis cycle . YFP, a recombinant protein, dissolve during mitosis and leads to increasing of noise in protein concentration. In contrast, droplets reform in most postmitotic cells and decrease the noise in protein concentration. These findings indicate that the RNA helicases play an important role in phase separation [DDXs globally promote phase separation in their ATP- and RNA-bound state. ATP hydrolysis induces the release of RNA clients from a DDX, results in the disassembly of RNA-containing membrane-less organelles . The conparation . The potRNA helicases are highly conserved enzymes important for RNA metabolism, which are involved in multiple steps of cell cycle regulation . RNA hel


Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs.The transcriptomes of IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that—if replicated in other species—will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria. Pseudomonas aeruginosa is a globally distributed opportunistic pathogen that thrives in aquatic habitats and can colonize the animate surfaces of plants, animals, and humans  offers the possibility of sequencing native RNA molecules . Direct 25–isolates .P. aeruginosa clone C, i.e., the cystic fibrosis clinical isolate NN2 and the environmental aquatic isolate SG17M   . P. aeruof 99.9%  (ENA accof 99.9% –36. Of t–sHsp20c  and the and clpG . HoweverStrains NN2 and SG17M share their core genomes and most regions of plasticity (RGPs), with a sequence diversity of just 0.01% consisting of 109 intergenic single nucleotide variants (SNVs), 464 synonymous SNVs, and 158 nonsynonymous SNVs. Most sequence diversity among the 2,338 SNVs resides within PAGI-2 genomic islands . Strain P. aeruginosa NN2 or SG17M grown in lysogeny broth (LB) at 37°C until mid-exponential (4 h) or early stationary (8 h) phase. After poly(A) tailing and library preparation, RNA sequencing was performed with MinION flow cells in the 3′-to-5′ direction. Three biological replicates were investigated on separate occasions. Only 4 out of a total of 143,693 total spike-in enolase control reads (0.003%) were spuriously antisense associated, indicating that the strand specificity of the P. aeruginosa RNA molecules had been retained during processing and sequencing.RNA was extracted from The number of available pores and generated reads varied widely during sequencing runs and between the individual flow cells . HoweverP = 0.002) (P = 0.002)  . Antisen= 0.002) . PrincipP. aeruginosa NN2 transcriptome at mid-exponential and early stationary phases also by cDNA Illumina sequencing. The RNA preparations were split into two aliquots, one of which was treated with terminator 5′ phosphate-dependent exonuclease (TEX)  , 41 to rThe paired comparisons  revealed that sense and antisense transcript levels were most similar between ONT and TEX and most divergent between TEX and 0-TEX. Hence, our experiments detected more differences in gene expression between the primary and the bulk transcriptomes derived from the same RNA preparation than between biological replicates that had been processed by different sequencing technologies on separate days. Apparently, our Nanopore protocol primarily yielded a primary transcriptome.Next, we compared the abundances of tRNAs in our samples because this class of RNA molecules has not received much attention in bacterial transcriptomics. A reason for the scarce literature may be the fact that both the Illumina and the ONT protocols do not detect aminoacylated tRNA molecules, because the 3′ end of the tRNA used for adapter ligation (Illumina) or polyadenylation (ONT) is blocked by the amino acid.P. aeruginosa PAO1  Lys-anticodon-TTT-pKLC102  extends into the plasmid attachment site of the genomic island pKLC102, which integrates into the 3′ end of the chromosomal tRNALys gene close to the pilA locus (−1 (The most abundant molecule in the tRNA pool was the transfer-messenger RNA (tmRNA) SsrA, known to play a key role in the rescue of ribosomes, stalled on a truncated mRNA and the tagging of the nascent peptides for degradation , 48. ConlA locus . pKLC102ocus , the overall abundance of uncharged sense tRNA transcripts significantly correlated with genomic codon usage, with average correlation coefficients of about 0.3 and 0.2 for experiments based on the Nanopore and Illumina platforms, respectively. However, the inspection of the individual associations uncovered a more complex picture. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing (In  missing  for the  missing .More than 90% of the antisense RNA molecules were complementary to a coding sequence. Only a few molecules were antisense tRNAs (see above) or matched with a complementary noncoding sequence other than rRNAs . The GentufB gene is flanked upstream by the triplet tRNATyr-tRNAGly-tRNAThr genes and downstream by the tRNATrp gene, followed by the genes secE, nusG, rplK, and rplA, encoding a protein cotranslocator, a transcription termination factor, and two ribosomal proteins. The majority of antisense RNA reads either covered the triple tRNA gene cluster or aligned to the gene contig tRNATrp gene-secE-nusG and parts thereof. A minority of >1-kb-long molecules mapped also to the other genes. This example shows a preponderance of conserved transcription and termination sites but also a large population of molecules with variable lengths and map positions. The next two examples demonstrate that this variability is not a technical artifact introduced during sample processing but reflects the natural variation of antisense RNA in P. aeruginosa. The second example . A few antisense RNA molecules mapped to other sequences of this region.To provide an impression about the spectrum of antisense RNA molecules in the clone C strains, we selected three genomic regions to which a comparably large number of antisense RNA reads had been mapped in all experiments  and 6. P example  and E de example , 49. Alm example  and F waNext, we wanted to explore whether the pools of mRNA and antisense RNA molecules were carrying diagnostic signatures that differentiate mid-exponential and stationary growth in NN2 and SG17M from each other. PCA indeed segregated the four experimental groups by growth phase in the first dimension and by strain in the second dimension whereby the biological replicates clustered together . Gene exprima facie from the sequence data. The only clear exceptions were the cell division genes ftsA and ftsZ. And indeed, the NN2 bacteria were longer and showed a broader size distribution than the SG17M bacteria , a population of longer asRNAs was sequenced that could be complementary to a contig of several genes and covered a broad range (a few hundred to thousands of bases)  molecules mapped to coding sequences consistent with the three previous reports that dealt with antisense RNAs in ruginosa , 52, 53.ruginosa  identifiruginosa  and 6. Tf bases) . The Kapplatform  and by dplatform . Since tDirect RNA sequencing was rewarding in the detection of RNA entities that are typically not covered by conventional cDNA-seq protocols. However, we observed several shortcomings of the current Nanopore RNA pipeline. First, base-calling was time-consuming and computationally intensive on central processing unit (CPU) hardware, though this was completely alleviated later after acquisition of graphics processing unit (GPU) hardware. Second, flow cells exhibited variable quality. Prior to starting the sequencing experiments, quality checks of flow cells were performed according to the protocol. Oxford Nanopore Technologies offers to replace any flow cells with less than 800 pores available for sequencing. However, our work plan required immediate sequencing of the processed samples, and flow cells should not be stored for more than 3 months. We hence initiated sequencing on flow cells with less than 800 pores. Consequently, some experiments were executed with poorly performing flow cells with a low number of available pores . HopefulThen direct RNA sequencing could become an integral component of the portfolio for bacterial transcriptomics and may even replace conventional cDNA-seq in many applications. We can envisage that direct RNA-seq could deliver unbiased, quantitative, and comprehensive strand-specific transcriptomes including meaningful information about RNA isoforms, antisense RNAs, operon structure, and transcription initiation and termination sites.P. aeruginosa clone C isolates NN2 and SG17M were stored in triplicate as glycerol stock cultures at −80°C. To assess the cell morphology of the strains, the bacteria were grown at 37°C in LB until mid-exponential and early stationary phases, precipitated, resuspended to a density of 107 bacteria/mL, and then immediately examined by light and electron microscopy at the Core Unit for Electron Microscopy (Jan Hegermann).The 600) of 0.05 and two uninoculated LB controls. The newly prepared bacterial suspensions were grown at 37°C on a shaker platform (150 rpm). After 4 h  and 8 h (early stationary phase), OD600 values of all replicates were obtained. At both time points, a subsample of each bacterial suspension (0.5 mL) was separately mixed with 1 mL of TRIzol reagent  for RNA extraction and subsequent Nanopore sequencing.Bacteria were cultured in 25 mL of LB overnight at 37°C on a shaker platform (150 rpm). Two uninoculated LB controls were processed in parallel. After 18 h, fresh LB suspensions  were set up with starting optical densities at 600 nm  buffer containing 1.5 mg/mL of lysozyme, and cells were lysed for 15 min at room temperature. Thereafter, RNA was prepared with the RNeasy minikit  following the protocol of the manufacturer. The RNA preparation was purified from contaminating DNA by two cycles of on-column DNase I digestion. RNA was eluted from the column with 2 × 75 μL of H2O. After addition of 2 μL of RNasin, the solution was stored at −80°C. The quality of the RNA preparation was checked with an Agilent 2100 BioAnalyzer .RNAprotect  was added to each aliquot after sampling and centrifuged for 10 min at 5,000 × Pairs of strand-specific cDNA libraries were prepared by the Eurofins Genomics Europe Sequencing Laboratory in Constance, Germany. One 0-TEX library was generated from bulk RNA for global transcriptome analysis, and the other library of enriched primary transcripts was generated from RNA that had been preincubated with terminator 5′ phosphate-dependent exonuclease (TEX) to remove 5′ monophosphorylated mRNA species , 41. Theg). The aqueous upper phase was isolated and mixed with isopropanol (500 μL) without pipetting but rather snapping against the tube, thereby avoiding RNA shearing into small fragments. The solution was centrifuged . The supernatant was discarded and the pellet was carefully resuspended in ethanol , without pipetting. The samples were centrifuged . The washing step with ethanol was repeated. The pellet was air dried (2 min) and resuspended in RNase-free water by snapping against the tube (50 μL). An additional bead-based purification step  including two further rounds of pellet washing with ethanol (75%) was incorporated. The purified RNA was resuspended in nuclease-free water (60 μL). The Qubit fluorometer and NanoDrop spectrophotometer were used for assessing RNA concentration and purity.The TRIzol suspension was vigorously shaken and incubated for 5 min at room temperature (RT) with regular tube inversions. Chloroform was added (200 μL). The mixture was incubated  and centrifuged  polymerase  kit. After the final incubation , an additional bead-based purification step including two further rounds of pellet washing with ethanol (75%) was performed with a final pellet resuspension in 12 μL of nuclease-free water. The Qubit fluorometer and NanoDrop spectrophotometer were used for assessing RNA concentration and purity, and the technical replicates were pooled to achieve a final poly(A)-tailed RNA concentration of 800 ng per sample in 9 μL of nuclease-free water.Bacterial RNA was poly(A) tailed with the Oxford Nanopore Technologies’ library preparation protocol for direct RNA sequencing (SQK-RNA002) was followed as recommended. Human enolase mRNA  was spiked into the bacterial RNA samples. The libraries (200 ng of RNA) were loaded onto MinION flow cells  and run for a minimum of 8 h. RNA was sequenced in the 3′-to-5′ direction.https://github.com/colindaven/guppy_on_slurm. Due to the intensive nature of base-calling, we strongly recommend using a GPU. A single Compute Unified Device Architecture (CUDA)-capable GPU is more energy efficient and far quicker than an entire compute cluster for this purpose, generating data in minutes instead of days.All computation was performed on the internal MHH HPC-seq 1000 core Ubuntu Linux compute cluster using software described at 2 fold change was applied to improve the fold change estimates and extract pairwise platform comparisons . The read quality was assessed with FASTQC 0.119  were obtained with the R function cpm of edgeR 3.34.0 . For short-read DNA sequencing, aliquots were directly loaded onto a COVARIS S220 Focused-ultrasonicator to induce DNA fragmentation. Afterwards, the DNA was purified with AMPure XP beads , and the NEBNext Ultra II DNA library prep kit E7645, E7103) protocol was used. The DNA library was sequenced on the Illumina NextSeq 550 platform , so that approximately 21.9 million (1.8 Gb) forward and 21.9 million (1.7 Gb) reverse reads were generated. For Nanopore sequencing, the DNA obtained after cell lysis underwent direct AMPure XP bead purification. Replicates were pooled to achieve DNA concentrations of 1 μg in 47 μL of nuclease-free water. Oxford Nanopore Technologies’ library preparation protocol SQK-LSK109 was followed as recommended, and the library was loaded onto a MinION flow cell (FLO-MIN106). About 19.3 million reads were generated (3.9 Gb) and base-called with Guppy. The read quality was assessed with FastQC, and adapters were removed with Trimmomatic 0.39 5, E7103 . Illuminonadales .https://github.com/mmpust/direct-RNAseq-2021). FAST5 files have been submitted to the public domain of the ENA (PRJEB46647). The complete reference genome of SG17M can be obtained from the National Center for Biotechnology Information .Coding scripts are publicly available from Github (


Penaeus chinensis) collected from a farm in China in 2016. Like most bunyaviruses, OWV1 particles were enveloped, spherical- to ovoid-shaped, and 80–115 nm in diameter. However, its genome was found to comprise four segments of (-)ssRNA. These included an L RNA segment  encoding an RNA-directed RNA polymerase (RdRp) of 2,052 aa, an M RNA segment  encoding a glycoprotein precursor (GPC) of 922 aa, an S1 RNA segment  encoding a nucleocapsid (N) protein of 243 aa, and an S2 RNA segment  encoding a putative non-structural (NSs2) protein of 401 aa. All the four OWV1 RNA segments have complementary terminal decanucleotides (5′-ACACAAAGAC and 3′-UGUGUUUCUG) identical to the genomic RNA segments of uukuviruses and similar to those of phleboviruses and tenuiviruses in the Phenuiviridae. Phylogenetic analyses revealed that the RdRp, GPC, and N proteins of OWV1 were closely related to Wēnzhōu shrimp virus 1 (WzSV-1) and Mourilyan virus (MoV) that infect black tiger shrimp (P. monodon). Phylogenetic analyses also suggested that OWV1 could be classified into a second, yet to be established, species of the Wenrivirus genus in the Phenuiviridae. These wenriviruses also clustered with Wenling crustacean virus 7 from shrimps and bunya-like brown spot virus from white-clawed crayfish. Of note there were no homologs of the NSs2 of OWV1 and MoV/WzSV-1 in GenBank, and whether other crustacean phenuiviruses also possess a similar S2 RNA segment warrants further investigation. In addition, we established a TaqMan probe-based reverse-transcription quantitative PCR method for detection of OWV1, and it was detected as 1.17 × 102—1.90 × 107 copies/ng-RNA in gills of 23 out of 32 P. chinensis samples without an obvious gross sign. However, the discovery of OWV1 highlights the expanding genomic diversity of bunyaviruses.Herein, we describe a novel bunyavirus, oriental wenrivirus 1 (OWV1), discovered in moribund oriental shrimp ( Bunyavirales order was established in 2016 to allow the taxonomic assignment of a plethora of new viruses discovered in diverse vertebrates, invertebrates, and plants. The number of members in the order has significantly increased in recent years, especially those discovered by next-generation sequencing, particularly meta-transcriptome  and kuruma shrimp (P. japonicus), but less frequently detected in banana shrimp (P. merguiensis) inhabiting eastern Australia  . It was ustralia ,b. Histo ganglia . In P. jrm ponds . Its patrm ponds .P. monodon and ridgetail prawn , respectively, collected from Wenzhou of Zhejiang Province, China (Cherax quadricarinatus) collected from Atherton Tableland in northern Australia  with genome composed of three RNA segments and Wēnzhōu shrimp virus 2 (WzSV-2) with two RNAs, were identified from tissue samples of e, China . Furthere, China . As for ustralia  and someustralia .P. chinensis sampled from a farm in China in 2016. Based on the evidence obtained in the present study, we suggest a name oriental wenrivirus 1 (OWV1), and also propose a binomial name Wenrivirus orientalis for the species to classify OWV1  from a hatchery in Shandong Province, China, and transported them alive  to the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences for use in bioassays. Within 2 weeks of being stocked across several aquaria, the shrimp displayed evidence of morbidity without typical clinical signs but suffered from ∼20% mortality from unknown causes. Cephalothorax tissues were sampled from the moribund shrimp to screen pathogens using transmission electron microscopy (TEM) and various PCR/RT-PCR methods. Total RNA and DNA were separately extracted from three pooled samples, and each pooled sample included five cephalothorax tissues. The molecular diagnosis of shrimp pathogens, including white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), Enterocytozoon hepatopenaei (EHP), and acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus (VpAHPND), was performed using RT-PCR or PCR as previously described  of the lymphoid organ, hepatopancreas, and gills were sampled and fixed in the TEM fixative  to screen any pathogens. Ultrathin sections were cut, mounted on collodion-coated grids, stained with aqueous uranyl acetate/lead citrate using standard procedures, and examined using TEM at the Equipment Center of the Medical College, Qingdao University.Small pieces . The pellets were resuspended in the TN buffer  and loaded onto a sucrose density gradient prepared by layering equal volumes of 20.0, 31.5, 43.0, 54.5, and 66.0% (w/v) sucrose and ultra-centrifuged in a P40ST rotor at 130,000 × g for 170 min at 4°C. The density gradients were fractionated. Aliquots of the band were diluted with TN buffer and ultra-centrifuged again in a P90AT rotor at 130,000 × g for 120 min at 4°C. The pellets obtained were then resuspended in a total volume of 1 mL TN buffer. Drops of the resuspended materials were placed unto grids, negatively stained with 2% phosphotungstic acid (PTA) (pH 6.5), and examined using TEM  operating at 80 kV.Approximately 3 g soft cephalothorax tissues excluding hepatopancreas were sampled from the moribund shrimp and homogenized in 10 mL TNEP  containing 200 μL protease inhibitor  using a TissueLyser (Qiagen). The homogenate was clarified by low-speed centrifugation, and the supernatant was then centrifuged at 10,000 × ® Ultra™ Directional RNA Library Prep Kit for Illumina® . Nest generation sequencing (NGS) was performed on the Illumina Hiseq platform, employing a 150 bp paired-end sequencing strategy. This step and the subsequent bioinformatics analyses were performed as described previously , and ribosomal RNA (rRNA) was depleted using a Ribo-zero™ rRNA Removal Kit . RNA sequencing libraries were generated from the rRNA-depleted RNA using NEBNexteviously .To obtain the full-length genome sequence, we designed PCR primers based on the consensus contigs assembled from the Illumina Hiseq reads . RNA wasBLASTx of the deduced RdRp, glycoprotein precursor (GPC), and nucleocapsid (N) protein sequences against the GenBank database was performed, and the top BLASTx hits to OWV1 were downloaded. Multiple sequence alignment was performed using MAFFT , and the2+, mutated MMLV reverse transcriptase, TOROIVD 5G DNA polymerase, RNase inhibitor, reaction buffer, and stabilizer, etc.) , 0.5 μM OWV1-S1-F and OWV1-S1-R primers each, 0.2 μM probe, and 1 μL RNA template. The amplification reaction was performed using a CFX96 Quantitative Fluorescence Instrument , and the reaction procedure was initiated at 52°C for 5 min and 95°C for 10 s, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s.We developed a reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) targeting the S1 fragment. The product fragment (71 bp) was amplified using the forward primer OWV1-S1-F (5′-CCG ACA TGG ATG CGT TCA-3′), the reverse primer OWV1-S1-R (5′-CAA GGC TGC AAC AAT GAC CTT-3′), and the TaqMan probe (5′-6FAM-CGC AGA CAT CCA GTT CCA GGG CTT T-TAMRA-3′). The TaqMan probe-based RT-qPCR for OWV1 (TaqMan-RTqPCR-OWV1) in a 20 μL mixture, containing 10 μL Probe 1-step RT-qPCR 5G Premix , infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), Enterocytozoon hepatopenaei (EHP), and acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus (VpAHPND). Among the three pooled samples, one sample tested positive for WSSV.DNA and RNA extracted from soft tissues of cephalothoraxes of the moribund P. chinensis identified spherical- to ovoid-shaped virions (80 nm∼115 nm in diameter) in the cytoplasm of the lymphoid cells , 48,574 mapped to the M RNA segment , and 8,038 mapped to the S RNA segment (coverage: 924 ± 477). To confirm the 3′ and 5′ terminal sequences of each RNA segment, we designed primers for Sanger sequencing to generate 5′- and 3′-RACE clones . Sanger E-value = 0; query coverage: 100%/100%), followed by Wenling crustacean virus 7  and bunya-like brown spot virus   and also a Bunya_RdRp superfamily domain cl20265 .BLASTp analyses revealed that the OWV1 RdRp  encoded by the L RNA segment   was mostge: 79%) . MoreoveE-value ≤ 4.34e-13) and the Phlebovirus_G2 superfamily domain cl09431  .The GPC glycoprotein (912 aa), encoded by the M RNA segment  of OWV1, possessed two transmembrane domains: the Phlebovirus_G1 superfamily domain cl06326  . These denvelope . BLASTp E-value ≤ 2e-99, coverage: 90%/92%), followed by BBSV   protein (243 aa) encoded by the S RNA segment  was most similar to those of WzSV-1/MoV  .However, it was reported that MoV contained an additional small (S2) RNA segment  encoding a putative non-structural protein  . BLASTx Wenrivirus together with WzSV-1/MoV  in inverse proportion to the threshold cycles (CT) with a slope of -4.118 and an intercept of 48.66. The established standard curve had a correlation coefficient of 0.994. Three parallel CT values of each sample are nearly the same, indicating that the intra-assay repeatability was reliable. We collected 32 P. chinensis samples from two markets, which had no obvious gross sign when sampling. After being tested with TaqMan-RTqPCR-OWV1, 23 samples were positive for OWV1 at 1.17 × 102—1.90 × 107 copies/ng-RNA of OWV1 in gills of P. chinensis.The TaqMan-RTqPCR-OWV1 method was established using OWV1 standard templates in nine concentrations from 1 × 10P. chinensis in China. The morphology and genome sequence of the novel virus revealed that it was mostly related to the wenrivirus MoV and its geographic variant WzSV-1. The difference in its genome sequence from the known virus species and the phylogenetic analyses of the RdRp sequences of OWV1 and other phenuiviruses support it to be a member of new virus species under the genus Wenrivirus. We then suggest naming the new virus as OWV1 from the common host name . Consecutively, we intend to prepare and submit an official taxonomic proposal to the ICTV to classify OWV1 as a member of a new species named Wenrivirus orientalis to fit recently adopted binominal species nomenclature  identical to the genomic RNA segments of uukuviruses and similar to those of phleboviruses and tenuiviruses in the Phenuiviridae. The conservation of 5′, - 3′ termini may be the characteristic of viruses whose genomes have multiple RNA segments. Thirdly, OWV1 lacks a variable GA-rich repeat sequence found in the S1 RNA segment of MoV/WzSV-1  that feeds on aquatic animals had a close relationship with Wenling crustacean virus 7 (Cyamus) and whale barnacles (genus Cryptolepas) (The hosts of the viruses of the family  viruses . The eco virus 7 . Further virus 7 . In additolepas) . TherefoP. chinensis cohort experiencing morbidity and mortality, it should be noted that co-infection of WSSV and OWV1 was found in this case. In particular, OWV1 of 1.17 × 102 - 1.90 × 107 copies/ng-RNA was detected in gills of P. chinensis without an obvious gross sign. 4 copies/ng-RNA (> 107 copies/μg-RNA in the paper) of MoV in lymphoid organs of challenged P. japonicus associated with an increase of lymphoid spheroids. It has been experimentally confirmed that MoV infection can cause gradually accumulating mortality in P. japonicus (P. monodon can tolerate a high-level MoV infection up to > 106 copies/ng-RNA (>109 copies/μg-RNA in the paper) in gills and that mortalities of the mid-crop mortality syndrome (MCMS) outbreaks are associated with the co-infection with gill-associated virus (GAV) rather than MoV. Therefore, whether OWV1 is responsible for or contributes to the pathology and the reduction in shrimp production clearly warrants further study.Crustaceans are one of the most important economic aquatic animals. The world exports of shrimp and prawn products exceeded 23 billion dollars, accounting for 16% of the global fishery commodity trades in 2018 . While Oaponicus . HoweverMK335503 , MK335504 , MK335505 , and MT040832 .The full-length genome of WvO has been deposited in GenBank under accession numbers: XD, WS, and JH designed the study and wrote the manuscript. YR, FM, CL, JCh, JS, and RW performed the biological experiments. XD, TH, JL, MS, and WS assembled preliminary sequences and analysis. QZ, PZ, CL, JCo, and KT discussed the results and modified the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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A recent meeting at Cold Spring Harbor Laboratory focused on emerging nucleic acid therapies and the essential academic research that has enabled them. The program encompassed everything from chemical innovations to preclinical and clinical progress, and provided a glimpse of the breakthroughs yet to come. With the rapid approval of the mRNA-based vaccine for emergency use in the fight against COVID-19 there is an increased public awareness in the potential of RNA-based medicines. Thus the recent Cold Spring Harbor Laboratory meeting on Nucleic Acid Therapies convening many of the world’s leaders could not have been more timely, or exciting.Beyond COVID-19, the delivery of mRNA vaccines against other diseases, including cancer, seems now poised to achieve great success.The importance of fundamental advances in chemistry and biology to development of cutting-edge pharmaceuticals was a key theme of the meeting. RNA being perhaps one of the least biologically stable of the macromolecules, it is uniquely versatile in its application to novel therapies. RNA can be modified in the base, backbone and sugar to increase target affinity, to thwart nuclease digestion and to maintain the drug’s chemical integrity once administered in vivo. Shulin Gao at Ionis Pharmaceuticals provided an overview of the progress made in RNA modifications and their impact on drug development since the late 1980s, when first generation chemistries—phosphorothioates (PS) modified with a sulfur on their phosphate backbone—were developed. Further innovations on this theme have led to significant improvements in therapies. For example, replacing the 2′-hydroxyl group on the ribose sugar of the PS backbone with a methoxyethoxy group improves target affinity and hence therapeutic activity. A talk from Muthiah (Mano) Manoharan at Alnylam Pharmaceuticals highlighted more recent developments, such as LICA  that improve tissue uptake and biodistribution, showing particular promise against liver diseases.An iconic example of fundamental research in RNA biology driving innovation in therapeutics is the development of CRISPR-Cas gene editing. Although already of great interest to researchers and pharmaceutical companies alike, the recent Nobel Prize in Chemistry awarded to Jennifer Doudna and Emmanuelle Charpentier for CRISPR technology has accelerated both research and drug development. Among the many highlights were three talks illustrating different avenues of current research. Samuel Sternberg at Columbia University presented a talk entitled, “Targeted DNA integration without double-strand breaks using CRISPR RNA-guided transposons,” describing a bacterially derived system—essentially an RNA-guided integrase enabling large-scale sequence changes without requiring double-strand DNA breaks and homologous recombination. Keith Gagnon and his group at Southern Illinois University focused on structure–function relationships in CRISPR-Cas12 and how varying the chemistry of the sgRNA pseudoknot affects the activity of this system. And finally, Briana Wilson at the University of Virginia demonstrated how blocking a specific cellular RNase stabilizes ultra-fast decaying small RNAs, thus promoting entry of small RNAs into the RNA-induced silencing complex (or RISC) as well as target repression. From the pharmaceuticals industry perspective, Christian Dombrowski and Kate Zhang from Intellia Therapeutics and Editas Medicine, respectively, provided insight into how companies are harnessing these discoveries. For example, cytosine base editing screening and optimization or engineering of AsCas12a to achieve high level on-target editing in hematopoietic stem cells, induced pluripotent stem cells, and natural killer (NK) cells, with promising results against sickle cell disease and antitumor activity.Fittingly, the program ended with exciting preclinical and clinical progress. Anastasia Khvorova at the University of Massachusetts Medical School gave a beautiful talk demonstrating the efficacy of RNAi targeting against Alzheimer’s disease, achieving results in the brains of experimental mice where the plaques associated with this disease were clearly abrogated. We also heard from Arthur A. Levin at Avidity Biosciences Inc., who in his talk entitled, “Antibody oligonucleotide conjugates—Progressing towards the clinic”, showed that by conjugating ASO to antibody, the pharmacokinetics in monkeys could be greatly improved with pronounced increase in tissue distribution and prolonged gene silencing activity in multiple tissues. And lest we forget about the most important recent development in RNA-based therapies, Philip Dormitzer from Pfizer gave the keynote fireside chat entitled “COVID-19 Vaccine Development”.Of course, a limitation to RNA-based therapies is that the targets are invariably within cells. Thus, much of the meeting focused on how to engineer RNA drugs that can overcome two main barriers to reaching its intended target within the cell. First, the RNA must be able to penetrate the cell membrane. On this point, Cosmin Mihai at Moderna, Inc. presented an elegant talk entitled, “Dissecting cytosolic mRNA delivery with single molecule resolution enables rational LNP design”, highlighting methods he developed to quantify mRNA in cells in order to evaluate lipids within the lipid nanoparticle (LNP) that might release more free RNA into the cytosol. Once inside the cell, the RNA needs to escape endosomal entrapment. Rudy Juliano—formerly at the University of North Carolina Chapel Hill—has now formed a company in the North Carolina Research Triangle Park called Initos Pharmaceuticals focused on exactly this problem. They aim to produce small molecular compounds that can be co-delivered with therapeutic nucleic acids and assist antisense and splice switching oligomers in endosomal escape.The astounding speed with which vaccines were produced to battle COVID-19 has no doubt re-energized the community of researchers working on RNA biology and therapies. The promise of this field lies in combining all of the technologies discussed above and knowledge yet to be uncovered to develop novel therapies. Beyond COVID-19, the delivery of mRNA vaccines against other diseases, including cancer, seems now poised to achieve great success. We hope that in the aftermath of the pandemic, research effort (and funding) for both basic research and clinical applications of RNA biology will continue to advance with the aim of tackling traditionally intractable or rare diseases for which patients at the moment have little hope.