Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays . Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions. SARS-SARS-CoV-2 has a positive-sense RNA genome of approximately 30 kb, and viral RNA synthesis occurs in the cytoplasm of an infected cell. During RNA synthesis, several processes occur including replication of the genome and transcription of a nested set of subgenomic messenger RNAs, which encode for the suite of viral proteins, as well as the genome for ORF1AB. During viral RNA synthesis, errors can occur including point mutation of single nucleic acids and recombination . RecombiSARS-CoV-2 has caused a worldwide pandemic and has severe economic and health implications for all affected countries. Given that mechanisms of both point mutation and recombination operate in coronaviruses, monitoring for these changes in the SARS-CoV-2 genome is essential not only for potential contact tracing but also for confidence in vaccine efficacy, which are generally based around the S protein. Experience from developing vaccines against the coronavirus infectious bronchitis virus (IBV), which arguably have been the most successful vaccines developed against coronaviruses so far, has suggested that differences of as little as 5% between the S1 sequences can result in poor cross-protection against different variants of IBV . DiffereAnecdotal data from diagnostic laboratories (at least based in the UK) early in the outbreak suggested that nucleic acid-based diagnostics to SARS-CoV-2 and complications around the sampling processes can lead to unreliable results and false negatives. The advantage of both laboratory  and fieln = 24) were recruited under the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) Clinical Characterisation Protocol (CCP) (https://isaric.net/ccp) by giving informed consent. ISARIC CCP was reviewed and approved by the national research ethics service, Oxford (13/SC/0149). Samples from clinical specimens were processed at CL3 in the Outbreak Response Laboratory at the University of Liverpool, Liverpool, UK.Patients  by spin-column procedure or Trizol LS , according to the manufacturer’s instructions. Total RNA was purified from SARS-CoV-2 infected Vero cells following AVL inactivation using the Qiagen RNA Mini Kit. Infection of Vero cells was conducted at Public Health England, Porton Down at CL3. RNA samples were treated with Turbo DNase (Invitrogen).Alignments generated from the NCBI reference sequence for SARS-CoV-2 NCBI (NC_045512.2) and published sequences on GISAID were used to identify conserved regions for primer design. Primers  were choSuperScript IV (Invitrogen) was used to generate single-strand cDNA using random primer mix . The primer sets were used to generate 30 amplicons from the cDNA. Subsequently, primers were pooled to generate six sets each containing five primer pairs. Reactions were performed using 2 × Q5-High Fidelity master mix (NEB) with 0.5 μM of each primer for the individual reactions and 0.1 μM of each primer for the pooled primers. The reaction conditions were as follows: denaturation at 98 °C for 30 sec followed by 35 to 40 cycles of 10 sec denaturation at 98 °C, 30 sec annealing at 66 °C and then 50 sec of extension at 72 °C. A final extension step was done for 2 min at 72 °C.Following amplification, PCR products were purified at a 1:1 ratio with AMPure XP beads . The library was prepared as per the sequencing by ligation protocol with native barcodes for multiplexing .https://github.com/connor-lab/ncov2019-artic-nf). Raw aligned BAM files were used as input to SVIM, to detect putative deletions. Resulting candidate deletions were filtered to accept any with at least 5 supporting reads and intersected with reference gene annotations to determine which genes intersected with the deletions. For assembly of the SARS-CoV-2 genomes, Minimap2 (v. 2.17-r941) was used to align fastq sequences to the coronavirus 2 isolate Wuhan-Hu-1 reference genome (NC_045512.2) using the -ax map-ont parameters. Samtools (v.1.10) was used to sort and index alignment files, and Picard (v.2.23.4) was used to mark duplicates. A custom script written in perl (v.5.26.2) was used to determine viral genome coverage, the viral consensus sequence and the presence of indels. Viral genome coverage was visualised in RStudio (v. 4.0.2). For the identification of deletions within the SARS-CoV-2 genome, ONT RSLA library reads were aligned to the coronavirus 2 isolate Wuhan-Hu-1 reference genome using Minimap2, as part of the Nextflow ARTIC analysis pipeline -infected Vero cells was used to confirm that the primers could generate amplicons. This RNA was used as a template for cDNA synthesis followed by PCR using the conserved primers. This generated 30 separate amplicons covering the SARS-CoV-2 genome, and the synthesis and expected size of these amplicons were confirmed using agarose gel electrophoresis B. These The ability of the amplicon-based approach to detect SARS-CoV-2 was initially evaluated on nasopharyngeal swabs collected from patients with COVID-19. The first patient (REMRQ0001) was admitted to the Royal Liverpool University Hospital on 23 February 2020. The patient was a member of staff from the Diamond Princes cruise ship that had an outbreak of SARS-CoV-2 leading to passengers developing COVID-19 ,13. The A final nasopharyngeal swab was taken from patient REMRQ0001 on 27 February. This latter swab was not used for a diagnostic assay but was used to evaluate the sensitivity of the amplicon-based system using RNA isolated from the sample. In this case, several amplicon products were identified, though fewer than on the previous sample (note that the brightness on the gel image was adjusted post-exposure to show these products more clearly) C. The prThe second patient (REMRQ0002) had onset of symptoms of COVID-19 on 22 February 2020 and was admitted to the Royal Liverpool University Hospital on 27 February 2020 after a diagnostic nasopharyngeal swab taken on 25 February tested positive for SARS-CoV-2. A repeat swab taken on the day of admission against tested positive for SARS-CoV-2. A further swab was taken for research the same day, and RNA isolated from this swab was used to evaluate the amplicon system. The amplicons were visualised by agarose gel electrophoresis C, confirIn order to increase capacity and reduce complexity , primer sets were pooled into six groups . These worf3a and orf7a, which are associated with interferon antagonism and would result in potential defective genomes being present.Recombination is a major mechanism of genetic change in coronaviruses, and this will manifest in insertions and deletions being present in the SARS-CoV-2 genome . For corThis work demonstrates that both amplicon-based detection and subsequent sequencing are feasible for identifying the SARS-CoV-2 genome or nucleic acid in samples from patients with COVID-19. The rationale is based on amplifying and sequencing longer fragments than comparative techniques, and our approach was named Rapid Sequencing Long Amplicons (RSLAs) to reflect this and its origins based on identifying MERS-CoV from viral subgenomic messenger RNAs present in clinical samples in Saudi Arabia. The amplicon system was evaluated first on RNA purified from cells infected with SARS-CoV-2 as a positive control and then on RNA purified from nasopharyngeal swabs from patients with COVID-19. We would note that this system best lends itself to identifying and sequencing SARS-CoV-2 from RNA that is of good quality. In general, the Ct value of these diagnostic leftover samples was not known. Different platforms are used in different settings and diagnostic laboratories in the UK, and there is no common uniformity. Interestingly, the RSLA system could identify nucleic acid corresponding to parts of the SARS-CoV-2 genome in a sample from a patient that had tested SARS-CoV-2 negative by an alternative diagnostic nucleic acid-based test performed on a separate sample. Provided that the primer binding sites remain conserved in the pathogen being tested, RT-qPCR is generally more sensitive for diagnostic purposes. In this case, diagnostic reagents for RT-qPCR can be reassessed based upon using sequencing as sentinel for these events. We would note that, based upon our comparison, multiple sites should be targeted within the genome to increase diagnostic sensitivity. Not all regions of the viral genome had equal sequence coverage, and this may be reflective of differential RNA stability in a clinical sample and the gradient of subgenomic messenger RNAs. Nevertheless, there was sufficient coverage to build consensus genomes.The non-multiplexed RSLA approach has been compared to other viral genome sequencing protocols such as ARTIC and viral bait capture, using the Illumina MiSeq platform, further demonstrating the feasibility and flexibility of this method. Although RSLA generates less depth in comparison to other methods, the breadth of coverage tends to be more even . RNA quaWhile most analysis of genome variation in viruses with RNA genomes is focused on nucleotide substitution, it is recombination in coronaviruses that is one of the big drivers of adaptation and change. A novel genotype of human coronavirus OC43 has been shown to emerge through recombination . Indeed,


The origins of life on Earth have been the subject of inquiry since the early days of philosophical thought and are still intensively investigated by the researchers around the world. One of the theories explaining the life emergence, that gained the most attention recently is the RNA World hypothesis, which assumes that life on Earth was sparked by replicating RNA chains. Since wet lab analysis is time-consuming, many mathematical and computational approaches have been proposed that try to explain the origins of life. Recently proposed one, based on the work by Takeuchi and Hogeweg, addresses the problem of interplay between RNA replicases and RNA parasitic species, which is crucial for understanding the first steps of prebiotic evolution. In this paper, the aforementioned model has been extended and modified by introducing RNA sequence (structure) information and mutation rate close to real one. It allowed to observe the simple evolution mechanisms, which could have led to the more complicated systems and eventually, to the formation of the first cells. The main goal of this study was to determine the conditions that allowed the spontaneous emergence and evolution of the prebiotic replicases equipped with simple functional domains within a large population. Here we show that polymerase ribozymes could have appeared randomly and then quickly started to copy themselves in order for the system to reach equilibrium. It has been shown that evolutionary selection works even in the simplest systems. According to the RNA World theory, early evolution of life involved RNA molecules both as genetic information carrier and catalyst (enzyme) . Strong in vitro selection, 24-3 polymerase ribozyme is the most proficient one , D is the diffusion constant and Brownian dynamics is used for diffusion in the simulation. Only single agents can diffuse, while complexes are assumed to be “heavier”, thus their diffusion is not explicitly performed. During diffusion, each agent is moved by a random vector, computed according to the following formula:onal one :w′→=2Δtint_radius—see Agents which are closer to each other than interaction radius (simulation parameter E) and it has to be in the folded state in order to be active . The second agent serves as a template (subscript T) and will be copied during replication, it can be either a parasite or a replicase. In order to step into the reaction, it has to be unfolded to ensure that the whole sequence is accessible during replication. Considering this, the probability of complex formation, when two agents meet, can be computed from the following formula:aE and lE are first agent’s (replicase) replication efficiency and its probability of being in the folded state, lT is the probability of being in the folded state for the second agent (template).Agent which initiated the complex formation is considered a replicase (subscript The replication procedure is as follows. The template is copied and new agent (with the same sequence) is put in the same position. Next, the mutation is performed, so the resultant sequence can be slightly different from it’s template. After replication, the complex dissociates.During dissociation both agents are “released”. They can move freely and their “aging” is resumed.The algorithm was implemented using C++ programming language and OpenMPI package.rank variable denotes the current thread id, whereas size is the total number of threads.The whole algorithm is presented below in the form of the pseudocode. Some purely implementational details have been omitted for simplicity. The code is executed by each thread separately. The command “synchronize” waits for all threads to execute and then the program execution can continue. The Algorithm 1: Main loop1forstep ← 1 tomax_stepsdo2  Diffusion and decay (Algorithm 2);3  synchronize;4  Simulate interactions (Algorithm 3);5  synchronize;6endAlgorithm 2: Diffusion and decay1 Make list list of thread’s agents ordered randomly;2whilelist not emptydo3  Take first agent A from list;4  ifA is complexedthen5   Decrease A replication time;6   ifA replication time is0 then7    Replicate A’s complex and perform mutation for newly created agent;8    Dissociate A’s complex;9   end10  end11  else12   Decrease A lifetime;13   ifA lifetime is0 then14    Remove agent A from simulation;15   end16   else17    Diffusion for agent A18   end19  end20endAlgorithm 3: Interactions of agents1 Make list list of thread’s agents ordered randomly;2whilelist not emptydo3  Take first agent A from list;4  ifA is not complexedthen5   Make list list2 of agent’s neighbours ordered randomly;6   ifSize of list2 is greater than simulation parameter neighthen7    Remove agent A from simulation;8   end9   else10    whilelist2 not emptydo11     Take first agent n from list2;12     ifn is not complexedthen13      reaction_probability = A.a * A.l * (1 − n.l);14      Take a uniformly distributed random number p from [0; 1);15      ifp < reaction_probabilitythen16       Form complex of A and n;17      end18     end19    end20   end21  end22enda, l and the changes in the simulated population size are presented on the charts. These values correspond to the average values taken from the whole simulated population in each simulation step.The main goal of the simulation was to analyze the behaviour of the large population of RNA molecules. Using multi-agent system described in the previous section, we conducted several simulations to check different conditions. We analyzed the case when the initial population consisted only of ideal replicases and the case when in the beginning of the simulation all RNA sequences were completely random and some of them happened to have accidentally proper replicase sequence. We also considered the scenario in which we checked how the additional information concerning the chemical stability of individual phosphodiester bonds connecting neighbouring nucleotides in the RNA strand influences the simulated population. Each time, the parameter values The analyzed system is considered alive if there is at least one RNA strand in the simulation area. Simulations were conducted until the system went extinct (there was no RNA strands anymore) or reached stability (nothing was changing over long time).Parameters used in simulations see  were mosl values for replicases and parasites (separately). The number of simulated steps was 10000, but after 8000 steps the system reached stability. Results are presented in Figs In this scenario, one hundred thousand ideal replicases were randomly placed in the simulation area. The goal was to check the stability of population consisting of replicases only and observe if parasites can appear and drive the whole system to extinction. During the simulation, multiple important values were measured, such as: the number of replicases and parasites, average replication efficiency and average l parameter is on average much lower for parasites . The replicase population shrinks significantly, in the same time parasties reach their peak and their number also dwindles a little bit . At the arasites , which ms stable .In this scenario, all RNA sequences were completely random. Some of them by accident had a proper replicase motif. This situation is much closer to the one, where first RNA molecules appeared randomly. Certainly, whether random sequences can kick-start evolution is an unsolved question. However, the results presented here show that if some replicases emerge from a random pool of RNA sequences, then their population can grow and take over the system, which can finally reach stability.The number of steps simulated was the same as in the previous scenario 10000. Results were presented in Figs Evolution of the system is very interesting. First, most of the parasites decayed, because they did not have any access to replicases. Population of replicases started to diminish as well, because the majority of them was too weak to sustain the tiny population . As a rel parameter  parameters on the simulated population. Hydrolysis rates of the phosphodiester bonds are presented in This simulation was carried out to check the influence of It is worth noting that cytosine and uracil were partially eliminated from the system (occurred much less frequently in the sequences). Despite of this, the results are almost the same as in the Scenario 1.In this scenario, simulations were carried out taking into account different values of parameters in order to check how they influence the results and the simulated population was smaller as compared to previous scenarios. The main goal was to show the parameters ranges where the system collapses versus survives. Two parameters were tested:d) - its value determines how long a single RNA molecule can live or how often one generation has to be replaced by the next one. This has a great influence on the speed of the evolution.base decay rate (D) - this parameter determines how important is the spacial aspect. The lower D is, the less homogenous the system becomes, because the diffusion is slower.diffusion constant  [In this paper, we have described the extended version of the parasite-replicase system implemented using multi-agent modelling approach (MAS). Similarly as the previous one , it can b) –40.a). Thus, only the RNA molecules carrying such a motif can act as RNA polymerase ribozymes (replicases), similarly as in grid Monte-Carlo model with a resolution at the individual molecules level presented in [Moreover, since it was postulated that the presence of specific domains dedicated to the certain functions were probably required in case of RNA replicases adaptive evolution , 57, 58,ented in . Contrarented in , where oIt is worth mentioning, that a similar approach was taken by Takeuchi and Hogeweg . They inIt is worth noting that our objective was not to discover the exact motif having certain sequence content, length and function, but to observe the mechanisms of evolution and to show that such motif could have emerged in the early prebiotic enviroment and contribute to evolutionary change (see Scenario 2). Thus, on account of our ignorance in this aspect, we decided to use the arbitrarily presumed sequence to characterize replicases, but in general, any other sequence could be chosen.In , apart fa (replicase efficiency) and l (fraction of the time spent by the RNA molecule in the folded state) being the stabilizing factor in the system. These assumptions are consistent with [a and l, can change during the simulations as a result of the mutations in the RNA sequence.Unfortunately, in the model of , the RNAent with , 31–33. ent with , agents The Scenarios 1-3 with evolving parasites and replicases show that the system is able to survive and allows for the stable coexistence for both quasispecies. The situation is similar, whether the simulation concerns the population of replicases only, where parasites appear due to the high mutation rate, or the population of the initial RNA sequences is completely random and some of them accidentally are equipped with the replicase functional motif. Since the RNA replicases are challenged by parasites and the ribozyme-derived parasites are unavoidable, in order to survive, they should tend to assemble together to promote the catalysis of their own replication. In our system, it is possible through the finite diffusion, which causes that replicases are located within close proximity of their relatives (agents with similar RNA sequences and parameters). Their limited dispersal allows for the preservation of strongly altruistic ribozymes, observe the kin selection and consequently, most likely overcome the parasite problem.d and diffusion constant D are crucial regarding the system survival and evolution. Base decay rate should have the optimal value, because if it is too low then the population is much smaller, but on the other hand if it is too high then the system goes extinct. Diffusion constant can seriously affect the system if it is close to 0, because it prevents the propagation of the replicases, so the population is unable to expand.Additionally, Scenario 4 was carried out in order to show the parameters ranges where the system collapses versus survives. The results of the simulations clearly show that the values of two parameters, namely base decay rate l parameter of replicases decreased quickly reaching very low level close to 0, while a parameter increased rapidly to the maximum level close to 1. In case of parasites, l parameter dropped slightly below 1. It means that, the replicases were definitely better recognized and favored by other replicases as templates, while parasites were rarely copied, which is also consistent with the results presented in [In , it was ented in , 60. Thea) drops in the simulations, but remains still high (over 0.8), while the probability of being in the folded state (l) is also reduced and stabilizes in case of both agents at a level below 0.3. It means that the selection favors better templates and good enzymes in the same time. Additionally, the l parameter was on average lower in case of parasites, which means that they evolved toward being good templates that are better recognized and easier copied than replicases.On the contrary, simulations conducted in this study show that it is possible to reconcile two conflicting goals at the same time, namely being a carrier of genetic information (good template) and catalyst (having a good replicase activity). It is noticeable that the average value of replication efficiency Click here for additional data file.S1 Fig(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.


However, the lack of RNA targeting activities limits the ability of CRISPR/Cas9 to combat RNA viruses. The recently identified class II type VI CRISPR/Cas effectors (Cas13) are RNA-targeting CRISPR enzymes that enable RNA cleavage in mammalian and plant cells. We sought to knockdown the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) directly by exploiting the CRISPR/Cas13b system. Effective mRNA cleavage by CRISPR/Cas13b-mediated CRISPR RNA (crRNA) targeting the ORF5 and ORF7 genes of PRRSV was observed. To address the need for uniform delivery of the Cas13b protein and crRNAs, an all-in-one system expressing Cas13b and duplexed crRNA cassettes was developed. Delivery of a single vector carrying double crRNAs enabled the simultaneous knockdown of two PRRSV genes. Transgenic MARC-145 cells stably expressing the Cas13b effector and crRNA mediated by lentiviral-based transduction showed a robust ability to splice the PRRSV genomic RNA and subgenomic RNAs; viral infection was almost completely abrogated by the combination of double crRNAs simultaneously targeting the ORF5 and ORF7 genes. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome  Vaccination is the primary strategy to prevent viral infections in hosts3. Unlike DNA viruses, the extremely high evolutionary rates of RNA viruses have led to the rapid emergence of variant virus strains5. Heterologous infections often cause vaccine failure. In addition, vaccines against many RNA viruses have not been successfully developed6. Following viral infection, antiviral drug treatments are the only clinical therapy to combat viruses. However, there are only a limited number of antivirals available targeting viruses that threaten public human health, such as HIV, HBV, HCV, herpesviruses and influenza viruses7. Although certain compounds have the potential to be developed as broad-spectrum antiviral agents, the relatively high cost limits their widespread clinical use in veterinary medicine, RNA viral infection causes significant economic loss in the livestock industry.RNA viruses remain a great threat to humans and animals around the world9. Porcine reproductive and respiratory syndrome virus (PRRSV), the aetiological agent of PRRS, is an enveloped single-stranded positive-sense RNA virus belonging to the family Arteriviridae10. Presently, PRRSV is taxonomically classified into the genus Porarterivirus12. The PRRSV genome is approximately 15 kb in length with at least 10 open reading frames (ORFs)13. PRRSV utilizes two distinct transcription mechanisms to express viral accessory and structural proteins14. The two-third 5′ terminal PRRSV genome contains two replicase-associated genes (ORF1a and ORF1b) encoding polyproteins that are further processed into 17 non-structural proteins (NSPs) responsible for PRRSV transcription and replication16. The PRRSV structural proteins are produced by a nested set of 3′ co-terminal subgenomic RNAs17. PRRSV consists of two highly heterologous species, namely, PRRSV-1 (formerly European genotype 1) and PRRSV-2 (formerly North American genotype 2)18. The PRRSV-1 species is classified into three subtypes (subtypes 1–3) and the PRRSV-2 species is further divided into typical and atypical strains21. The PRRSV-1 Lena strain (subtype 3) and PRRSV-2 atypical strains have been described as highly pathogenic PRRSV strains characterized by high fever and high mortality rates in pigs of all ages22. PRRSV can suppress the host immune system, which may allow secondary/opportunistic pathogens to establish infections, resulting in more severe and chronic diseases23. Due to extensive antigenic variability and immunomodulatory properties of the virus, control of PRRSV has proven to be a challenge throughout the world. Therefore, the development of alternative and effective treatment strategies is urgently needed.Porcine reproductive and respiratory syndrome (PRRS) has been one of the most important infectious viral diseases in swine worldwide for almost three decades25. The CRISPR-Cas systems form two major classes, namely, class 1 and class 2 which employ multiple Cas proteins and a single large Cas protein for nucleic acid interference respectively26. Considering the simple effector structure, class 2 CRISPR-Cas systems have been widely engineered as genome-editing tools28. Class 2 consists of three types  and can be further divided into several subtypes31. Among them, type II Cas9 is the most commonly studied CRISPR effector for double-stranded DNA (dsDNA) engineering33. CRISPR RNA (crRNA) leads the Cas9 protein to recognize and bind target sites and then results in a double-strand break (DSB) approximately 3 bp upstream of the protospacer adjacent motif (PAM) through the RuvC and HNH nuclease domains35. The DSBs can be subsequently repaired by the homology-directed repair (HDR) pathway which requires a donor template or the non-homologous end joining (NHEJ) pathway in the absence of a homologous template within the target DNA for genome editing purposes37.The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems provide adaptive immunity to bacteria and archaea, enabling organisms to respond to and eliminate invading foreign genetic elements40. In contrast to Cas9, the HNH and RuvC domains do not exist in Cas13 proteins. The dual higher eukaryotes and prokaryotes nucleotide (HEPN)-binding endoRNase domains of Cas13 mediate the precise cleavage of targeted transcripts44. The splicing activity varies in different Cas13 subtypes and orthologues. By comparison of 21 Cas13a, 15 Cas13b and 7 Cas13c orthologues, the Cas13b orthologue from Prevotella sp. P5–125 (PspCas13b) proved to be the most robust and specific for RNA knockdown in mammalian cells40. In addition, the most recently discovered Cas13d was more efficient than PspCas13b38. Importantly, type VI enzyme-mediated RNA cleavage shows no PAM preference in eukaryotes, allowing flexible design of potential targeting sites40. In bacteria, Cas13 exhibits nonspecific cleavage of any transcripts near the target and activates programmed cell death to limit bacteriophage infections43. Fortunately, such collateral RNA degradation activity is not present in mammalian and plant cells, allowing specific RNA targeting40. Furthermore, Cas13 exhibits a high specificity of RNA interference activity relative to RNA interference (RNAi), as no off-target effects were detectable in eukaryotic cells40. In addition to RNA knockdown capability, fusion of catalytically inactive Cas13 (dCas13) to an RNA-editing enzyme retained the RNA-binding activity and enabled specific RNA editing in mammalian cells40. These advantages of type VI CRISPR systems have the potential to be developed as a platform to combat RNA viruses by targeting and degrading viral RNA in mammalian cells.Recently, type VI Cas effectors (Cas13) were identified as RNA-targeting CRISPR enzymes enabling RNA knockdown in mammalian and plant cells displaying high specificity and targeting flexibilityHere, we sought to investigate the possibility of adopting the CRISPR/Cas13b system for interference against PRRSV RNA in eukaryotic cells. To this end, we designed multiple specific crRNAs targeting the PRRSV essential genes ORF5 and ORF7. Our study revealed that the CRISPR/Cas13b catalytic activities resulted in interference with PRRSV gene transcription and expression. Furthermore, a novel all-in-one CRISPR/Cas13b delivery system that enabled cells to be transfected with the Cas13b effector and duplex-targeting guides simultaneously was established. The CRISPR/Cas13b system enabled an almost complete knockdown of PRRSV genomic and subgenomic RNAs to eradicate viral infections in lentiviral-mediated transgenic MARC-145 cells expressing Cas13b and duplexed crRNAs. Our study indicated the potential of using the CRISPR/Cas13b system as a novel therapeutic strategy by directly targeting the viral genes of RNA viruses.45, was selected as the target. A PRRSV ORF7 gene fused with an eGFP reporter plasmid was generated, and a set of guide RNA targeting sequences was designed and then inserted into the CRISPR/Cas13b guide RNA backbones  protein, which is the main component of the viral capsid encapsulating viral RNA and is involved in the regulation of host cell processesnes Fig. . To assenes Fig. . At 48 hnes Fig. . Flow cynes Fig. . To furtnes Fig. . Conversnes Fig. , demonstnes Fig. , indicatin vivo46, for screening. Six and four regions were selected to target the ORF7 and ORF5 genes, respectively  and the classic PRRSV (strain 01NP1) were identical (data not shown), we determined the effect of the CRISPR/Cas13b system on the viral infectivity of both strains. Initially, the cells were infected with the HP-PRRSV strain 10PL01 and the classic strain 01NP1 at an MOI of 0.1. The culture medium was harvested every 12 h to determine the viral growth kinetics. The MARC-145 cells containing Cas13b and specific PRRSV crRNAs displayed high levels of virus resistance Fig. , Fig. S4in vitro.To investigate CRISPR/Cas13b knockdown activities on PRRSV genomic RNA of the 10PL01 strain, we performed qRT-PCR to detect NSP9 gene, which does not exist in any subgenomic RNAs encoding viral structural proteins. Consistent with the viral growth kinetic studies, compared with the two control cell lines, MARC-145 cells expressing crRNA 5-2 and 7-1 showed strong PRRSV genomic RNA splicing with reductions of 93.8% and 97.0%, respectively Fig. . Combini49. Multiple dsDNA viruses have been subjected to antiviral CRISPR/Cas9 targeting in vitro and in vivo, including EBV, HSV-1, HCMV50, HBV51 and HPV52. During a viral infection, there are two main strategies to inhibit the virus by using CRISPR/Cas9 technology: (i) targeting host genes that are essential for virus infection and (ii) targeting viral DNA to directly eliminate viral replication53. The major obstacle for the application of the CRISPR system in RNA virus infections is that most functionally characterized systems target dsDNA rather than RNA. Therefore, previous studies selected the host essential genes CCR5 and CXCR4, which are the chemokine co-receptors responsible for viral entry for HIV infection, as targets56. Alternatively, by selectively disrupting HIV proviral DNA integrated into the host genome, some groups have demonstrated that targeting HIV long terminal repeats (LTRs) or essential viral genes is an effective approach to profoundly suppress HIV production in vitro62 and in vivo63. However, there are no dsDNA intermediates in the majority of RNA viruses, and host essential genes are the only choice for CRISPR/Cas9 targeting, including for TGEV64, IAV65, CSFV66, FMDV67 and BVDV68. A recent study described the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of exon 7, which encodes SRCR5 of the CD163 gene responsible for binding to GP4 to mediate viral entry. These pigs showed complete resistance to type 1 PRRSV infections69. Although the size, stature and other morphological features of these pigs were comparable to those of wild-type pigs, the long-term impact of the disruption of functional cellular proteins remains uncertain.CRISPR/Cas technologies are powerful tools for gene therapy70. In plants, the Cas13a orthologue from Leptotrichia shahii (LshCas13a) showed approximately a 50% RNA interference with turnip mosaic virus (TuMV)70. The mild inhibitory efficacy is probably due to the less robust RNA knockdown ability of Cas13a than that of Cas13b. During the revision process of this manuscript, the same group further demonstrated that LwaCas13a, PspCas13b and CasRx variants (Cas13d) could mediate high interference activities against plant RNA viruses. Moreover, Cas13d exhibited the most robust interference ability in plants71. Meanwhile, several studies had focused on implementing CRISPR/Cas13-based strategies on RNA viruses, including the SARS-CoV-2 virus73.The Cas13 enzyme is the first CRISPR-based system to enable precise RNA targeting and editing. The antiviral activities of type VI genotype have been determined in plants44, whereas PFS constraints are absent in mammalian cells40. CRISPR/Cas13 tolerates one mismatch between target RNA and crRNA, and the existence of more than two mismatches decreases the cleavage activity43. The optimal Cas13b targeting condition is not fully understood. A previous study indicated that the Cas13 binding and cutting efficiency were related to the secondary structure of the target RNA75. The low homology between PRRSV-1 and PRRSV-2 makes it difficult to design crRNAs targeting both PRRSV species within the ORF5 or ORF7 gene  has been shown to affect Cas13b activityene Fig. . Althougene Fig. , this doOne of the major challenges for implementing the CRISPR/Cas-based genome engineering system is the efficient and simultaneous delivery of the Cas protein and guide RNAs. The Cas protein and guide RNA have been introduced into cells mostly via independent plasmids by co-transfection. Co-transfection of multiple plasmids leads to variable expression of CRISPR components in each cell. To overcome this problem, we developed a single CRISPR/Cas13b-duplexed crRNA delivery system that efficiently expressed the Cas13b effector and dual or multiple mature guide RNAs. In addition, this system included an eGFP marker to indicate Cas13b expression. Although no accumulative effect of Cas13b was detected by targeting one gene with two or three guide RNAs, the established all-in-one platform enabled the simultaneous knockdown of ORF5 and ORF7 mRNAs by incorporating two corresponding guide protospacer sequences. Although the delivery efficiency of the all-in-one system still needs to be improved, the results showed the potential of CRISPR/Cas13-based multiplex RNA inference in mammalian cells.78. In vitro, MARC-145 cells, derived from the African green monkey kidney cell line MA-104, display high susceptibility to PRRSV infection and are commonly used for PRRSV studies79. However, MARC-145 cells with a low chance of transfection  did not sufficiently support CRISPR/Cas13 system delivery by an established all-in-one vector. To address this limitation, we employed an all-in-one lentiviral platform for the generation of stably expressing CRISPR/Cas13 MARC-145 cell lines. We observed high levels of PRRSV viral genomic knockdown in cells carrying a single crRNA. Notably, transgenic cells showed almost complete cleavage activities against genomic RNA and abrogated PRRSV infection with duplexed crRNAs targeting ORF5 and ORF7. The marked improvement in RNA splicing ability could be attributed to the lentiviral-mediated increase in robust and sustained expression of Cas13b and crRNAs compared to that observed after transient transfection. In addition, the establishment of CRISPR/Cas13 system expression prior to PRRSV infection favours the elimination of viral RNA. Nidovirus subgenomic RNAs are synthesized from the genomic RNA template by the discontinuous transcription mechanism80. The significant reduction in all PRRSV subgenomic RNAs in transgenic cells was mostly due to robust cleavage of viral genomic RNA. Since the splicing levels of PRRSV genomic RNA in the two single-crRNA cell lines were comparable, we further assessed the Cas13b cleavage specificity on PRRSV subgenomic RNAs. As the subgenomic RNAs shared the 3′ co-terminus, the 7-1 targeting region existed in all six subgenomic RNAs, whereas the 5-2 targeting region was absent in the two latter subgenomic RNAs. The qRT-PCR results showed significant differences that were detected in only subgenomic RNA 6 and 7 between the Cas13b-crRNA-5-2 and Cas13-crRNA-7-1 cell lines, demonstrating the specific viral subgenomic RNA knockdown ability of Cas13b. Since previous studies clearly indicated that no detectable off-target effects were caused by Cas1340, off-target effects were not addressed in the current study.PRRSV has a specific cell tropismin vivo. Although the lentivirus-mediated CRISPR/Cas delivery system prevented RNA virus infections in transgenic cells, the integration of exogenous genes into the host genome, especially in livestock animals, is a cause for concern in terms of food safety and public perception. The adeno-associated virus (AAV) system could be a potential alternative, but the large size of Cas effectors limits the packaging efficiency in AAV. The most recently identified type VI-D CRISPR effector Cas13d, the smallest class II Cas protein in mammalian cells, can be packaged into the AAV system and be potentially delivered into natural viral hosts38. CRISPR/Cas9-mediated dsDNA virus targeting may introduce mutations, insertions or deletions at guide RNA targeting sites leading to virus variants that might be more pathogenic or resistant to CRISPR/Cas9-directed disruption81. Although virus escape mutants can be restricted by simultaneously targeting multiple sites, this is an obstacle to CRISPR/Cas9 application in antiviral therapy53. For CRISPR/Cas13, the latest research demonstrated that no crRNA target site mutations were observed following Cas13 cleavage over a course of 48 h, suggesting a low likelihood of production of mutations by Cas13 targeting73. However, further work is needed to comprehensively assess the possibility of introducing virus variants by CRISPR/Cas13 cleavage in the long term. Once the formation of virus escape mutants becomes unavoidable, how many target sites (or genes) will be required to limit virus variants and prevent virus evolution. PRRSV comprises several structural proteins and NSPs. In our study, ORF 5 and ORF 7 have been proven to be ideal targets for CRISPR/Cas13 cleavage. However, other genes especially the genes coding NSPs that are essential for PRRSV transcription and replication will need to be investigated in the future.The current study represents the first application of CRISPR/Cas13 against mammalian RNA viruses in eukaryotic cells. However, there are still several challenges to implementing the CRISPR/Cas13 system to interfere with RNA virus infections in vitro, and can potentially be a potent therapeutic antiviral strategy. The impact of the current study will not be limited to PRRSV, as the results have a wider significance, and this study provides the foundation for studies about other RNA viruses infecting humans and animals.Overall, the prospect of developing a CRISPR/Cas13 system to directly knockdown viral RNA represents an exciting avenue in antiviral therapy. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome 82 and typical strain 01NP1 were kindly provided by Chulalongkorn University Veterinary Diagnostic Laboratory  and propagated in MARC-145 cells using DMEM supplemented with 2% FBS. The viral titers were calculated by the Reed-Muench method83 and expressed as a 50% tissue culture infective dose (TCID50). PspCas13b fused with the HIV Rev gene NES expression vector (Addgene 103862) and PspCas13b guide RNA expression backbone with the U6 promoter (Addgene 103854) were generous gifts from Feng Zhang40. The eGFP reporter vector pEGFP-N1 was obtained from Clontech (Japan). The RFP657 reporter vector pTagRFP657-N1 was a gift from Vladislav Verkhusha (Addgene 31959)84. The lentiviral packaging plasmid pCMV-dR8.2 dvpr (Addgene 8455) and the VSV-G envelope plasmid pMD2.G (Addgene 12259) were gifts from Sheila A. Stewart85 and Didier Trono, respectively.HEK293T, HEK293FT and MARC-145 cells were obtained from the American Tissue Collection Center  and cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% foetal bovine serum  and 1% penicillin-streptomycin (Gibco). PRRSV-2 atypical strain 10PL01BsmB I restriction sites with different overhangs were introduced upstream of the EF-1α promoter to facilitate Golden Gate cloning of the guide RNA component cassettes. Next, Cas13b guide RNA backbones were modified by the introduction of BsmB I restriction sites with various overhangs. To generate the lentiviral transfer gene plasmid, the all-in-one vector was further modified by replacing eGFP with the puromycin gene and introducing the HIV genes 5′LTR, WPRE, cPPT/CTS and RRE. All primers are reported in Table To generate the PRRSV GP5-eGFP, N-eGFP, GP5-RFP657 and N-RFP657 reporter constructs, PRRSV viral RNA was purified from PRRSV-infected MARC-145 cells, and cDNA was synthesized. Subsequently, the ORF5 and ORF7 genes were amplified by specific primers and inserted into the pEGFP-N1 and pTagRFP657-N1 vectors, respectively. To construct individual ORF5 and ORF7 reporters, the CMV promoter and ORF5 or ORF7 gene were inserted into the PUC19 vector. For the Cas13b expression plasmid, the EF-1α promoter and PspCas13b fused with the HIV Rev gene NES and an HA tag were cloned into the PUC19 vector. To establish a delivery vector carrying the Cas13b protein and streamline duplexed guide RNA cassettes, the eGFP reporter gene was amplified from the pEGFP-N1 vector and inserted downstream of Cas13b-NES-HA linked by a T2A skipping peptide. Two E. coli cells , the plasmids were prepared by a Plasmid Midiprep Kit .The sense and antisense guide RNA oligonucleotides were synthesized Table  with ove4 HEK293T cells per well were plated into 24-well plates 16 h prior to transfection to ensure 60–70% confluency at the time of transfection. Transient transfections were performed using Lipofectamine 3000 (Invitrogen). For triple-plasmid transfection, cells were transfected with 250 ng of Cas13b vector, 500 ng of guide RNA plasmid and 250 ng of eGFP reporter plasmid. For dual-plasmid transfection, cells were transfected with 500 ng of all-in-one Cas13b-gRNA plasmid along with 500 ng RFP657 reporter plasmid.To assess PspCas13b activities in eukaryotic cells, approximately 1 × 10To determine the expression of reporter genes after CRISPR/Cas13b activity along with guide RNA targeting, transfected HEK293T cells were fixed in 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Subsequently, Fluoroshield mounting medium with DAPI  was dropped onto the cells, and imaging was performed by a Olympus IX73 inverted microscope with cellSens Dimensions imaging software .TM Select  and washed once with cold PBS by centrifugation for 5 min at 1,000 × g and 4 °C. The supernatant was removed by aspiration, and the cells were resuspended in 500 μl of FACS buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide). The cells were analysed using a Cytomics FC 500 MPL flow cytometry system . The data were analysed using MXP software . To select MARC-145 cells stably expressing Cas13b after infection with lentivirus, harvested cells were fixed in 4% paraformaldehyde and permeabilized with ice-cold 90% methanol. After washing, cells were stained with a 1:50 dilution of an HA tag antibody (Thermo Fisher). For secondary staining, a 1:100 dilution of goat anti-mouse IgG1-FITC was added to cells and incubated in the dark. For each measurement, at least 10,000 events were gated.At 48 h post-transfection, cells were dissociated by TrypLETM Total RNA Kit  with DNase treatment following the manufacturer’s instructions. The quality of the RNA was determined by a NanoDrop 1000 . The first-strand cDNA was synthesized by M-MuLV reverse transcriptase . Quantitative PCR was performed with Luna® Universal qPCR Master Mix  using a StepOnePlusTM Real-Time PCR System . The amplification reaction consisted of an initial denaturation at 95 °C for 1 min, followed by 45 cycles of denaturation at 95 °C for 15 s and extension at 60 °C for 30 s. The primers used for amplification are shown in Table t method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control for normalization.Total RNA from cultured cells was purified by the GenUPHEK293FT cells were plated into 10 mm plates and transfected with 5 µg of the constructed lentiviral transfer plasmid, 1.25 µg of pMD 2. G and 2.5 µg of pCMV-d8.2 dvpr by Lipofectamine 3000 according to the manufacturer’s instructions. At 6 h post-transfection, the packaging medium was replaced. Viral supernatants were collected 24 h and 52 h after medium replacement, pooled and clarified through a 45 μm PVDF filter (Millipore). The supernatants were then concentrated by Amicon Ultra-15 centrifugal filter units. For lentiviral transduction, MARC-145 cells were transduced with concentrated lentiviral supernatants supplemented with 8 μg/ml polybrene (Millipore). At 72 h post-infection, cells were screened via puromycin (6 μg/ml) treatment. After 3 weeks of selection, single clones were isolated by limiting the dilution. Successfully transduced cells expressing Cas13b and the corresponding guide RNA were detected by flow cytometry and PCR, respectively.For the virus inhibition assay, MARC-145 and parental MARC-145 cells with stable expression of CRISPR/Cas13b-gRNA were cultured overnight in a 12-well plate and then infected with PRRSV 10PL01 or 01NP1 at various MOIs. After 1 h of incubation at 37°C, non-adherent viruses were aspirated, and cells were replenished with DMEM containing 2% FBS. Cell supernatants were collected at different time points, and virus titers were determined. Cells were scraped from the wells in PBS and subjected to qRT-PCR to determine viral genomic and subgenomic RNA levels.All experiments were performed three times. Statistical significance was calculated using two-way ANOVA to determine the differences between the CRISPR/Cas13-targeted group and the control group. All statistical analyses were performed using GraphPad Prism software .Supplementary materials.


Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses , and other respiratory disease-causing viruses . Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 10 Among the coronaviruses, the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have caused threats to public health in 2002 and 2012 . By the betacoronavirus, with a sequence homology of 80–89% with coronaviruses discovered in humans, bats, and other wild animals  [SARS-CoV-2 falls into the genus ike CoV) ,20,22. Dike CoV) . Owing tThe impediment of further spread and transmission of this disease requires rapid and reliable identification, especially in areas with limited laboratory capacity. Concurrently, in hospitals, the identification of the causative agents of recurrent acute respiratory infections requires routine and confirmatory diagnosis through quantitative real-time polymerase chain reaction (qRT-PCR). In recent years, the loop-mediated isothermal amplification (LAMP) method that includes an exponential amplification of specific nucleic acid sequences at a constant temperature, has been widely utilized for the rapid detection of virus-specific genes ,27. The 2 RNA copies which is close to qRT-PCR. Furthermore, by combining the amplification process with colorimetric detection, the assay is suitable for rapid and simple diagnosis within poorly equipped primary hospitals and laboratories, and in situations where an urgent diagnosis is needed.In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay has been developed for the detection of the SARS-CoV-2 nucleocapsid (N) gene through colorimetric visualization. The study was aimed to develop a rapid, simple and sensitive molecular detection assay for the novel coronavirus that can differentiate it from other currently circulating human coronaviruses, including OC43, 229E, NL64, and MERS-CoV as well as other respiratory viruses. This method enables the detection of SARS-CoV-2 within 30 min, excluding RNA extraction, with the detection limit of 10www.gisaid.org). The sequences were aligned using CLC Genomics Workbench 10.0.1 . A conserved region was identified in the target N gene sequence  that was used as a template for the designing of the RT-LAMP primers.The RT-LAMP primers were designed for the detection of SARS-CoV-2 based upon the already available sequences in Global Initiative on Sharing All Influenza Data (GISAID)  programme based upon the published sequence of the N gene. Primer sets included two external primers (forward outer primer F3 and backward outer primer B3), two internal primers (forward inner primer FIP and backward inner primer BIP), and two loop primers (backward loop primer LB and forward loop primer LF). All primers were synthesized by Bionics, Inc. (Republic of Korea). in vitro transcription. The details of sequence and primers used for the RNA synthesis are provided in Supplementary Table 2. The artificial SARS-CoV-2 and SARS-CoV RNA was synthesized using MEGAscript® Kits  following the manufacturer’s instructions. RNA was recovered through lithium chloride precipitation and stored at −80 °C till further use. For quantification of RNA transcript, RNA copy number using EndMemo DNA/RNA Copy number calculator (http://endmemo.com/bio/dnacopynum.php) was calculated, quantitated using Qubit RNA HS Assay Kit  following the manufacturer’s instructions, and the end-point diluted RNA was confirmed through qRT-PCR.Initially, primers containing T7 RNA Polymerase promoter were designed using BetaCoV/Wuhan-Hu-1/2019  sequence for SARS-CoV-2 and SARS-CoV/HKU-39849  sequence, and outsourced from Bionics, Inc (Republic of Korea). The T7-flagged PCR product was then synthesized for 2. Intact viral RNA was extracted using RNeasy mini kit  followed the manufacturer’s instructions. The viral RNA was stored at −80 °C till further use. The complete experimental work with SARS-CoV-2 was conducted in Enhanced Biosafety Level 3 (BL-3+) facility at Chungbuk National University as approved by the Korea Center for Disease Control.Moreover, BetaCoV/Korea/NMC01/2020 and BetaCoV/Korea/NMC02/2020 viruses were propagated and passaged twice in Vero cells. Vero cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (FBS) and 1% antibiotics, and incubated at 37 °C in 5% COTo optimize the sensitivity and specificity of RT-LAMP detection, different primer concentrations  were tested. For a 10 μL RT-LAMP reaction, a master mix solution was prepared containing of WarmStart® Colorimetric LAMP 2X Master Mix  (5 μL), F3 and B3 primers (1 μL each), FIP and BIP primers (1 μL each), and LF and LB primers (1 μL each). The optimized final concentrations of all primers are represented in 11 to 1 × 100) synthesized viral RNAs in the RT-LAMP. Concomitantly, same samples were subjected to qRT-PCR according to the shared protocol developed by the National Institute of Infectious Diseases of Japan, which showed one of the highest sensitivities in the comparison study [The RNA template (2 μL) (extracted from virus and/or synthesized RNA) was added to the master mix and the RT-LAMP reaction was performed at 65 °C. Detection limit determination was performed by processing tenfold serially diluted  using the determined limit concentration of viral RNA. Ten repeats of the RT-LAMP reaction were performed using the determined viral RNA concentrations with incubations of 30 and 60 min. For comparison, the detection limit of qRT-PCR was also determined by performing ten repeats of the reaction using 10−9 and processed for parallel testing with RT-LAMP assay and qRT-PCR. The serially diluted RNA (2 μL) was mixed with the optimized master mix and subjected to RT-LAMP assay as described previously [The sensitivity of the developed RT-LAMP assay to detect SARS-CoV-2 was confirmed using the cell propagated cultures of SARS-CoV-2 that were grown and passaged twice in Vero cells. This intact viral RNA was ten-fold serially diluted to 10eviously .To determine the specificity of the optimized RT-LAMP assay for targeting SARS-CoV-2, the assay was tested across three-panel sets of different RNA samples that were extracted from patients’ nasal swabs and/or cell-propagated isolates of respiratory disease-causing agents. The RT-LAMP was cross-tested against a panel set of RNA samples of related coronaviruses, which includes human coronavirus 229E (229E), human coronavirus NL63 (NL63), human coronavirus OC43 (OC43), and Middle East Respiratory Syndrome coronavirus (MERS-CoV) tested using the developed RT-LAMP assay. The optimized RT-LAMP assay was also tested against a panel of human infecting and avian influenza viruses. The RNA samples of A/California/04/2009 (H1N1pdm), A/Perth/16/2009 (H3N2), B/Brisbane/60/2008 (Victoria lineage), and B/Phuket/3073/2013 (Yamagata lineage); highly pathogenic avian influenza viruses, including H5N1, H5N6, H5N8, and H7N9; and low pathogenic avian influenza viruses, including H2, H4∼H12 were also tested for any reactivity . FurtherFourteen nasal swabs collected from COVID-19 patients in the National Medical Center, Republic of Korea were processed for viral RNA extraction. Purified RNA was used for the clinical evaluation of the RT-LAMP assay in comparison to the qRT-PCR method as described above. An agreement between the two tests was also evaluated using Cohen’s Kappa.Clinical samples were collected from the National Medical Center, South Korea. Samples were collected under the approved guidelines and relevant regulations. The Ethics Committee and Institutional Review Board of National Medical Center (IRB no. H-2002-111-002) approved all experimental procedures. All experiments were performed following the approved guidelines.in vitro transcribed 2 μL of 1 ng/mL RNA (1.2 × 107 number of copies) of the SARS-CoV-2 N gene. A successful RT-LAMP reaction results in a colorimetric reaction which includes a change in the colour of phenol red pH indicator from pink to yellow  was used as the template in both the RT-LAMP   RNA copies across ten reactions (qRT-PCR mean Ct=37.43) (Supplementary Table 4).To optimize the RT-LAMP, and determine its limit of detection, a ten-fold serial dilution of the RNA transcript (1 × 103 copies of RNA per reaction to be observed at both time points (across ten repetitions). For 102 copies per reaction, the positive detection rate was 90% for 30 min and 100% for 60 min (across ten repetitions). Moreover, during the ten repetitions, the RT-LAMP was observed to possess an equal detection rate of 30% for detecting 101 RNA copies at both the time points  higher detection rate for the two intact viral RNA samples. The mean Ct values observed (across three repetitions) are indicated in The sensitivity of the RT-LAMP assay was tested to detect the intact viral RNA that was extracted from the cell culture supernatants of two isolates from COVID-19 patients. The sensitivity was observed to be ten-fold lower than that of the qRT-PCR, which was similar to that of the RT-LAMP assay performed using the synthesized RNA. Both assays were able to detect SARS-CoV-2 in a reaction in which intact viral templates were used  for 30 and 60 min incubation  were subjected to qRT-PCR which demonstrated a ten-fold higher detection rate in agreement with the RT-LAMP results using the synthesized RNA as template (Supplementary Table 4).The detection limit of RT-LAMP was determined using ten repetitions of serially diluted templates , MERS-CoV, influenza viruses, and other respiratory disease-causing viruses. No amplification was observed in RT-LAMP assay for any of the viral RNA panel of related coronaviruses  protein gene were utilized. In general, the gene encoding N protein was found to have the most abundant expression of subgenomic mRNA (sgmRNA) during infection . The corin silico analysis, the cross-reactivity between SARS-CoV-2 and SARS-CoV and/or Bat SARS-like CoV would be low based upon the sequence length of the RT-LAMP primer binding regions (10.4-12.3%) . In addition, the RT-LAMP assay using the synthesized RNA of SARS-CoV N gene showed positive amplification with 107 RNA copies per reaction (across three repetitions). This result shows 10,000-fold lower cross reactivity compared to that of SARS-CoV-2 N gene (102 RNA copies) . Furthermore, using Cohens Kappa, the level of agreement between qRT-PCR and RT-LAMP was revealed to be “almost perfect agreement”  [The limit of detection revealed that the developed assay is highly capable of detection and nearly as sensitive as qRT-PCR. We have demonstrated that the RT-LAMP assay is specific for SARS-CoV-2 and it possesses rare cross-reactivity with other viruses that manifest similar respiratory disease. Moreover, this assay can discriminate currently co-circulating common human coronaviruses and MERS-CoV. The data presented supports that the RT-LAMP is as specific as qRT-PCR in detecting SARS-CoV-2. In  0.8261) . This suGenerally, RT-LAMP methodology is regarded as new generation diagnostics . HoweverIn summary, we have developed an RT-LAMP assay that can be of use in clinical laboratories in support of the preliminary detection of SARS-CoV-2 in suspected patients especially during the testing of an abundant number of samples. With high sensitivity and specificity, this assay has a simple methodology, low cost, and is time efficient. Further, a single-tube isothermal colorimetric method does not require any expensive equipment. With the declaration of public health of international concerns against SARS-CoV-2, our method would significantly contribute to the public health responses and disease control of countries in providing preliminary counter detection measures, especially to those with limited laboratory capacities.


Proteins so far identified are members of the hnRNP family , as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, TDP‐43, MVP, LIN28, SRP9/14, QKI, and TERT. We describe the RBPs based on protein domain features, current knowledge on the association with human diseases, recognition of RNA consensus motifs, and the need to clarify the functional significance in different cellular contexts. We also summarize data on previously identified RBP inhibitor small molecules that could also be introduced in EV research as potential modulators of vesicular RNA sorting.Extracellular vesicles (EVs) are heterogeneous membranous particles released from the cells through different biogenetic and secretory mechanisms. We now conceive EVs as shuttles mediating cellular communication, carrying a variety of molecules resulting from intracellular homeostatic mechanisms. The RNA is a widely detected cargo and, impressively, a recognized functional intermediate that elects EVs as modulators of cancer cell phenotypes, determinants of disease spreading, cell surrogates in regenerative medicine, and a source for non‐invasive molecular diagnostics. The mechanistic elucidation of the intracellular events responsible for the engagement of RNA into EVs will significantly improve the comprehension and possibly the prediction of EV “quality” in association with cell physiology. Interestingly, the application of multidisciplinary approaches, including biochemical as well as cell‐based and computational strategies, is increasingly revealing an active RNA‐packaging process implicating RNA‐binding proteins (RBPs) in the sorting of coding and non‐coding RNAs. In this review, we provide a comprehensive view of RBPs recently emerging as part of the EV biology, considering the scenarios where:  EVs can originate from multi‐vesicular bodies (MVBs), giving rise to vesicles referred to as exosomes, or from the plasma membrane budding, in this case referred to as microvesicles (MVs) or ectosomes . The expansion of our knowledge on the different classes of RNAs populating the EV cargo, including coding and non‐coding species  results from the recent broad applicability of sensitive RNA detection technologies and computational tools. However, we are still far from predicting a specific RNA sorting into bulk or sub‐populations of EVs according to the cell physiology.The RNA is part of the EV cargo and the focus of many functions ascribed to EVs. Currently, the Vesiclepedia database includes more than 27,000 entries for mRNAs and more than 10,000 entries for non‐coding RNAs ((i) individual RBPs were detected in EVs along with their substrate RNAs, (ii) RBPs were detected in EVs but the substrate RNA targets are inferred only, and (iii) transcripts found in EVs which harboured sequence motifs mirroring the activity of RBPs.Very recently, the activity of competitive RNA‐binding proteins is emerging as a crucial determinant in diversifying the enrichment of selected transcripts into EVs. Interestingly, the application of biochemical, cell‐based, and computational approaches revealed in several models the enrichment of transcript sequences, defining ‘RNA motifs’, recurrently found in EVs and validated as consensus substrate for RNA‐binding proteins (RBPs). In this review, we provide a comprehensive view of the current knowledge about RBPs described as part of the EV biology. We included the scenarios where Irrespective of the methodologies used to isolate EVs, we highlight the protein domain features of RBPs detected into EVs Figure , the preHere, we describe members of the hnRNP family , as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, MVP, LIN28, SRP9/14, QKI, and TERT RNA‐binding proteins.2Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of 20 major and minor proteins in humans with differential RNA‐binding capacities . The protein was found responsible for direct interaction with a GGAG motif present in the 3′‐end of miRNAs  enriched in vesicles. Notably, the relevant activity of hnRNPA2B1 was associated with its sumoylation status  within exosomes. Lei et al.,  demonstrGiven the multiple roles of hnRNPA2B1 in disease, some screening campaigns identified small molecules proposed to interact with the RNA‐binding activity of the protein. MO‐460, an analogue of the natural product (R)‐(‐)moracin‐O, suppresses the accumulation in Hep3B cells of HIF‐1α (Hypoxia‐inducible factor‐1α), involved in tumour cell adaptation to hypoxic conditions. MO‐460 binds to the C‐terminal glycine‐rich domain of hnRNPA2B1, inhibiting its subsequent binding to the 3'‐untranslated region of HIF‐1α mRNA , also known as hnRNPG, as an ARTS‐1 (aminopeptidase regulator of TNFR1 shedding)‐associated protein, is involved in two mechanisms of TNFR1 secretion: the constitutive release through exosome‐like vesicles and the IL‐1β‐mediated inducible proteolytic cleavage of TNFR1 ectodomains. The RBMX RNAi reduces, in fact, TNFR1 release through both mechanisms  and healthy prostatic cells , chronic hepatitis B (CHB), and liver cirrhosis (LC) patients, resulting to significantly higher levels in patients compared to control groups. Also, high exosomal hnRNPH1 mRNA levels were associated with worse overall survival (OS) in HCC patients  suppression of exosome biogenesis and release  progression. The overexpression of lncRNA 91H was also observed in exosomes from HCT‐8, HCT‐116 cell lines, and in patients’ serum. Mass spectrometry analysis revealed a physical interaction between hnRNPK and lncRNA 91, further confirmed by RNA interference  has been described as a secreted factor in extracellular vesicles. The presence of SYNCRIP in hepatocellular exosomes was linked to the control of specific miRNAs , the single‐stranded motif GGGG, as well as either single‐ or double‐stranded forms of the motifs CACC and CATC. The YBX1 A/P domain interacts with SRp30c, p53, and cyclin D1, whereas it's CSD interacts with AKT kinase and E3 ubiquitin ligase. The CTD is involved in protein homodimerization. YBX1 directly interacts with proliferating cell nuclear antigen (PCNA), mutS homolog 2 (MSH2), X‐ray repair cross complementing 5 (XRCC5), and DNA ligase III, clearly indicating its involvement in DNA repair  is a RNA‐binding protein composed of an A/P rich domain , a cold shock domain (CSD), and a long C‐terminal domain with alternating positively and negatively charged amino acids , antisense TASRs, 3ʹUTR‐derived RNAs, and tRNA‐derived short RNAs (tsRNAs and tRFs)  ‐induced human endothelial progenitor cell (EPC)‐derived exosomes The high expression level of YBX1 is a poor prognostic marker in patients with breast cancer  is a member of the embryonic lethal abnormal vision (ELAV) family of RNA‐binding proteins  in the C‐terminal region seems implicated to poly‐A recognition and protein oligomerization  demonstrated an association between CAFs oxaliplatin resistance and the horizontal transfer of CCAL  lncRNA from neighbouring cancer cells , mediating gene‐silencing effects on their RNA targets  and stress granules  proteins to form the RISC loading complex, it processes precursor miRNAs into mature miRNAs  is a member of the insulin‐like growth 2 proteins, a family of RBPs conserved from insects to mammals  and associated with poor prognosis (in lung adenocarcinoma) at earlier onset: more than 3‐fold increased IGF2BP1 expression can be detected in 70% of NSCLCs where it positively correlates with a 30% reduction in tumour‐specific 5‐years survival after surgery  compared to exosomes derived from less aggressive cells (Panc02) AGGGG) and the downregulated (top enriched motif: AUGACGUA) genes in the transcriptomes which could implicate a direct or indirect sorting mechanism in EVs upon IGF2BP1 depletion , also known as RKHD2, BM‐013or RNF194, is a member of an evolutionarily conserved family of four RNA‐binding E3 ubiquitin ligase, known as MEX3 proteins, initially identified in MEX3C1, the longest isoform with 659 amino acid residues, was found overexpressed in cells of the innate immune system and in different types of tumours, such as breast, liver, and bladder cancer, where it displayed oncogenic properties by regulating lipid metabolism ‐(D/E), and allows the annexins to shuttle between water‐soluble and membrane compartments in response to fluctuations in calcium concentration. This property suggests an ability to influence membrane‐related events in response to the cell's homeostasis Hajjar, . StructuANXA2 is found in the cytoplasm as a monomer or in the cell surface as a heterotetramer consisting of two ANXA2 monomers bridged non‐covalently to an S100A10 dimer ‐Interacting protein X (ALIX) is one of the established protein markers enriched in exosomes  , discovered in 1973, is a highly conserved RBP and one of the major nucleolar phosphoproteins in eukaryotic cells , engineered by fusion of the novel human anti‐NCL scFv 4LB5 and a human pancreatic RNase, on exosomes from derivatives of colorectal cancer cells  is a predominantly nuclear RNA‐binding protein that belongs to the FET FUS, EWSR1, TAF15) protein family together with EWS and TAF15RBPs. FUS contains an N‐terminal domain with a glutamine‐glycine‐serine‐tyrosine‐rich (QGSY‐rich) region, a highly conserved RRM domain, a zinc finger motif, and multiple RGG amino acid repeats at the C‐terminal  of the multimeric vault ribonucleoproteins  upon MVP silencing. An RNA‐pull‐down assay validated the role of MVP on exosomes derived from HEK293 cells transfected with biotinylated full‐length MVP, whose RNA was later extracted and quantified  was able to downregulate the expression of MVP in A549 cells  and LIN28B. In vertebrates, these ribonucleoproteins share a high sequence identity (73%) and three conserved structural regions composed of an N‐terminal cold shock domain (CSD) and two knuckle‐type cysteine‐cysteine‐histidine‐cysteine (CCHC) zinc finger (ZnF) domains GAU sequence on RNA targets is necessary for their recognition by the CSD domain , where the function of let‐7 as tumour suppressor against RAS or MYC is lost  is a cytoplasmic ribonucleoprotein complex, which in eukaryotes consists of six polypeptides  and a 300‐nucleotide 7S RNA (RN7SL1)  as potential SRP9/14 inhibitors in a hepatocellular carcinoma mouse model. After SBPS treatment at different doses, the examination of the serum proteins revealed reduced SRP9/14 as compared to its levels in tumour‐bearing mice  and part of an evolutionarily conserved class of RNA‐binding proteins called STAR  proteins  are mainly expressed in the central nervous system (CNS), glial cells, oligodendrocytes, and neuronal progenitors  are spliced‐proteins encoded by the quaking gene , surrounded by QUA1 and QUA2 domains , mimicking the environment of hypoxia/reoxygenation (H/R) injury. They found an enrichment of miR‐208a and miR‐208b miRNA. Since QKI is known to inhibit the apoptosis of cardiomyocytes (CMs) and an in silico analysis predicted the binding site of miR‐208a and miR‐208b to 3’UTR of QKI, the authors tested the effect of this RNA‐RBP interaction by exposing hypoxic cardiomyocyte to EVs. Notably, the EV‐mediated transfer of miR‐208a and miR‐208b suppressed the protective influence of QKI on apoptosis, suggesting QKI as a target to regulate CM apoptosis  and the Inserted Hairpin Element 1 (IH1), independently from the C‐terminal active site   The neural sphingomyelinase 2 (nSMase2)‐dependent pathway; (ii) The sumoylated hnRNPs‐dependent pathway; (iii) The 3′ miRNA sequence‐dependent pathway; iv) The AGO2/miRISC‐related pathway. Besides the relative dosage/equilibrium of single miRNAs that could be indirectly co‐regulated by messenger RNAs and potentially described by the competing endogenous RNA (ceRNA) model , main RBP interactors, RNA motifs, and includes identified RBP small molecule inhibitors. Some of these inhibitors could represent potential tools that could be successfully used as complementary approaches to study an RNA‐packaging effect into EVs.The authors declare no conflicts of interest.


Such a service has been touted as being a useful, convenient, and safe means to reduce the risk of exposure to infection sources of the novel coronavirus. Nevertheless, this distribution method may still pose a potential risk of spreading the disease.Social distancing and a shelter-in-place order are among the measures implemented to effectively prevent the spread of coronavirus disease–2019 (COVID-19).2 This has raised a concern that food delivery has a great potential of contributing to the spread of the disease.3 While more and more people adhere to the shelter-in-place order, delivery workers are fulfilling customer orders. This has suddenly spurred them to the frontlines of the COVID-19 pandemic.4 The likelihood that delivery workers (1) have direct contact with novel coronavirus–infected customers without ever experiencing symptoms and (2) may subsequently act as a presymptomatic transmitter unwittingly passing the novel coronavirus to their healthy customers, coworkers, or families should be taken into consideration  or credit card payment method. In developing countries, digital payment or credit card payment is encouraged to limit contact with delivery workers.4.Discarding of packaging. Customers should discard the packaging as soon as possible and wash hands immediately after.During this time of pandemic, delivery workers are at higher risk of the novel coronavirus infection and potentially become a “spreader.” Here are practices to mitigate those risks:


Session types describe patterns of interaction on communicating channels. Traditional session types include a form of choice whereby servers offer a collection of options, of which each client picks exactly one. This sort of choice constitutes a particular case of separated choice: offering on one side, selecting on the other. We introduce mixed choices in the context of session types and argue that they increase the flexibility of program development at the same time that they reduce the number of synchronisation primitives to exactly one. We present a type system incorporating subtyping and prove preservation and absence of runtime errors for well-typed processes. We further show that classical  sessions can be faithfully and tightly embedded in mixed choices. Finally, we discuss algorithmic type checking and a runtime system built on top of a conventional (choice-less) message-passing architecture.


The data presented in this article are related to the research article entitled “Effects of carboxymethylation, acidic treatment, hydroxypropylation and enzymatic hydrolysis combined with heating on structural and physicochemical properties of palm kernel expeller dietary fibres.” This article describes the effects of carboxymethylation, acidic treatment, hydroxypropylation and enzymatic hydrolysis combined with heating on the structural and physicochemical properties of palm kernel expeller dietary fibres (PKEDF). Our data is made publicly available to the potential re-use of palm kernel expeller in food and other industries. Moreover, this dataset provides a reference about how to improve physicochemical and functional properties of dietary fiber. The data provide the potential re-use of palm kernel expeller in the food or other industries.The data provide information on how to improve some functional properties of palm kernel expeller dietary fiber.This data may serve as a reference for other works to develop other dietary fiber resource unutilized.1The 2In the current study, effects of acidic treatment, carboxymethylation, hydroxypropylation and dual enzyme hydrolysis combination with heating on structral and physicochemical properties of palm kernel expeller dietary fibres (PKEDF) were studied. Firstly, PKEDF was prepared from defatted palm kernel expeller with α-amylase, alcalase and glucoamylase. Then PKEDF was modifed by acidic treatment (PKEDF-A), carboxymethylation (PKEDF-C), hydroxypropylation (PKEDF -H) and dual enzyme hydrolysis combination with heating (PKEDF-HE), respctively. The chemical composition, particle size distribution, color, structural and phsicochemical properties incuding water swelling capacity, oil holding capacity, emulsifying capacity, emulsion stability and α-amylase activity inhibition ration were studied , 5, 6.2.12O) , and then 0.3 g of hemicellulase and 0.45 g of cellulase were added. The mixture was adjusted to pH 4.5 and incubated at 50 °C for 3 h. Afterwards the mixture was incubated in boiling water for 10 min, and then cooled and filtered. The residue was collected and dried at 45 °C for 4 h to obtain PKEDF treated by heating with enzymatic hydrolysis (PKEDF-HE).PKEDF (50 g) was heated at 121 °C for 45 min. The heated PKEDF was suspended in deionized water (dH2.22O and dried at 60 °C for 4 h. Then PKEDF treated by acidic (PKEDF-A) was obtained 1 mol/L NaOH  at 60 °C for 2 h, and then treated by 1 mol/L HCl at 60 °C for 30 min. Afterwards, the mixture was neutralized and filtrated, the residue was washed with dH2.3g for 15 min. The precipitate was collected, washed with anhydrous ethanol and dried at 45 °C for 4 h to obtain carboxymethylated PKEDF (PKEDF-C) PKEDF (8 g) were suspended in 80 mL of ethanol . The suspension was gently stirred at room temperature (RT) for 30 min. Then 50 mL of NaOH (0.676 mol/L) were gradually added with vigorously stirring at 35 °C for 60 min. Afterwards 9.7 mL of chloroacetic acid (3.38 mol/L) pre-treated with NaOH (3.38 mol/L) were added and gently stirred at 35 °C for 30 min. Then the mixture was incubated at 53 °C for 3.5 h, and cooled to RT, neutralized to pH 7.0 with acetic acid and centrifuged at 4000 2.42O and then mixed with 50 mL of Na2SO4 (20 mg/mL). The mixture was adjusted to pH 11.0 and 2 mL of propylene oxide were added. The mixture was stirred (180 r/min) at 40 °C for 24 h, and then filtrated and the residue was collected and dried at 45 °C for 8 h 5 g of PKEDF were suspended in 50 mL of dH2.5The scanning electron microscopy (SEM), Fourier-transformed infrared spectroscopy, and X-ray diffraction (XRD) analysis of DFs were performed using the same procedure as described by Zheng & Li The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


As an important regulatory mechanism at the posttranscriptional level in metazoans, adenosine deaminase acting on RNA (ADAR)-induced A-to-I RNA editing modification of double-stranded RNA has been widely detected and reported. Editing may lead to non-synonymous amino acid mutations, RNA secondary structure alterations, pre-mRNA processing changes, and microRNA-mRNA redirection, thereby affecting multiple cellular processes and functions. In recent years, researchers have successfully developed several bioinformatics software tools and pipelines to identify RNA editing sites. However, there are still no widely accepted editing site standards due to the variety of parallel optimization and RNA high-seq protocols and programs. It is also challenging to identify RNA editing by normal protocols in tumor samples due to the high DNA mutation rate. Numerous RNA editing sites have been reported to be located in non-coding regions and can affect the biosynthesis of ncRNAs, including miRNAs and circular RNAs. Predicting the function of RNA editing sites located in non-coding regions and ncRNAs is significantly difficult. In this review, we aim to provide a better understanding of bioinformatics strategies for human cancer A-to-I RNA editing identification and briefly discuss recent advances in related areas, such as the oncogenic and tumor suppressive effects of RNA editing. In mammals, ADAR-induced adenine to inosine (A-to-I) is a widespread primary type of RNA editing . As aden−/− embryonic phenotype because MDA5 is responsible for distinguishing and helping remove exogenous dsRNA, except for ADAR-edited dsRNA  (https://darned.ucc.ie/) (http://rnaedit.com/) (http://srv00.recas.ba.infn.it/atlas/claire.html)  , DARNED ucc.ie/) , RADAR (it.com/) , and CLAre.html) . There are.html) , while are.html) . ncRNAs re.html) , and mulre.html) . Abnormare.html) . As a spre.html) . Due to re.html) –34.+ cells   . In addim cells) . The ideIt is difficult to predict the function of RNA editing sites located in non-coding regions owing to their diversity and interactions with various ncRNAs. Moreover, except for typical RNA editing sites  that can be intuitively selected according to their locations, many other potentially functional RNA editing sites residing in non-coding regions still need to be explored and validated . This prThis review outlines the existing bioinformatics strategies for identifying editing sites in tumors, which can be used for further experimental verification of downstream effects and clinical relevance. Simultaneously, we provide some suggestions for ncRNA editing research and the potential application of ADAR inhibitors in the treatment of cancer.In 1991, the first A-to-I RNA editing report was published when researchers detected RNA editing events on GluR mRNA . At the Editing site calling is a complicated process involving many aspects and requiring appropriate optimization and high accuracy in each step. Therefore, researchers have summarized some functionally integrated pipelines –59. CurrAs mentioned above, the two popular strategies share a common calling strategy that includes the following four steps: (1) preprocessing sequencing data, (2) sequencing read mapping, (3) RNA editing calling, and (4) RNA editing site annotation. When analyzing RNA editing levels, studies usually employ certain common mapping tools, such as BWA , Bowtie Sequencing errors, DNA mutations, and a lack of a suitable SNP database will result in false-positive results that affect RNA editing detection. These sequencing errors are mainly caused by reverse transcription, homopolymers, low-quality sequences, etc. An important factor resulting in sequencing errors is RNA modification, such as m1A and m6A. The reverse transcriptase will likely misidentify modified nucleotides as other types of nucleotides  [e.g., mvia a novel SNP-free algorithm  to measure the global editing levels in different samples. Erez Y. Levanon and Eli Eisenberg et al. provided the Alu editing index (AEI) to measure the global editing levels in different samples. The AEI ratio weighted by A-to-G mismatches within Alu repeats relative to the total number of adenosines within Alu elements represents the average Alu editing levels, indirectly showing the overall RNA editing levels , 98.de novo identification of these unknown RNA editing sites is necessary.For the identification of RNA editing in tumor samples with both DNA-seq and RNA-seq data, we suggest BWA (DNA and RNA-seq) or BWA (DNA-seq) + STAR (RNA-seq) for mapping the sequencing data and REDITools for RNA editing calling. Maria et al. compared some commonly used alignment tools, including BWA, GSNAP, HISAT, and STAR, and RNA identification tools, such as RNAEditor, GIREMI, REDItools, RES, and JACUSA, and analyzed the ability of these tools to identify RNA editing . In theiWe suggest selecting unique molecular identifiers (UMIs) when building RNA-seq libraries, which will bring many advantages : PCR mutBased on tens of thousands of potential RNA editing sites reported from bioinformatics methods, many experimentally validated A-to-I editing sites and their regulatory mechanisms have been demonstrated. As shown in https://ncrnaome.osumc.edu/miredibase/) (for miRNA) (http://bioinfo.life.hust.edu.cn/LNCediting/) (for lncRNA)   and LNCe lncRNA) . Several lncRNA) . The ove lncRNA) . (3) In  lncRNA) . (4) In  lncRNA) . (5) Zip lncRNA) . (6) Hep lncRNA) .Tumor and adjacent tissue samples are suitable subjects for studying RNA editing—a large number of non-cancer-specific RNA editing sites can be excluded by comparing RNA editing in cancer and adjacent tissue. The investigation range of essential RNA editing sites affecting cancer occurrence and development can be effectively narrowed by comparing the editing levels in cancer and adjacent tissue. Most cancers are accompanied by detailed clinical data, from which the characteristics of RNA editing in different cancers can be summarized and the range of key targets can be further streamlined.ADAR1&2 expression is different in various cancers. ADAR1 expression increases in most cancer types, such as breast invasive carcinoma and liver hepatocellular carcinoma, but decreases in a few cancers, such as kidney chromophobe. Some researchers found that the expression of ADAR1 and ADAR2 in the same cancer can be totally opposite. For example, some studies show that ADAR1 is a potential tumor enhancer with high levels, and ADAR2 is recognized as a tumor suppressor in HCC , 108.The role of specific RNA editing level changes mediated by different ADAR enzymes in tumors has been partially discovered. To further determine which RNA editing site is closely related to tumor development and maintenance, researchers can select paired samples from cancer patients and identify these tumor-related RNA editing sites using statistical methods such as Fisher’s exact test and Wilcoxon rank sum and signed rank test. Studies have shown that certain site editing levels are greatly associated with patient survival . Therefovia Sanger sequencing , that effectively calls and annotates human A-to-I RNA editing events, predicting their downstream effects on pre-mRNA alternative splicing and miRNA-3’ UTR targeting via human high-seq data  in mouse spinal motor neurons and human SH-SY5Y cells, and circGRIA2 editing level alteration is a potential marker for early serum diagnosis of amyotrophic lateral sclerosis (ALS) since it can be secreted out of the cell . They clSince the ability of ADARs to deaminate has also been applied to the field of gene editing, they also have great potential in cancer therapy , 136. NeIt has been found that some chemically synthesized small-molecule drugs can inhibit the expression of ADAR1. Ding et al. reported that 8-chloro-adenosine can inhibit the ADAR1/p53 pathway, inhibiting the proliferation of breast cancer . ErythroTo date, great efforts have been made to develop computational methods alongside the advancement of sequencing technologies to detect RNA editing events, and millions of editing sites have been reported, allowing researchers to gain growing information on different tissues. However, many aspects could be optimized to explore tissue-specific editing levels in the future. In addition, the advent of third-generation long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore brings about more facilities for editing calling since it will theoretically circumvent the current technical bottlenecks, such as PCR errors and hyperediting read loss , 147. OnConceptualization: YZ and GS. Writing—original draft preparation: YZ, HW, and SC. Writing—review and editing: all authors. Visualization and table collection: HW and JW. Funding acquisition: YZ and GS. All authors contributed to the article and approved the submitted version.This research was funded by National Natural Science Foundation of China, grant number 31502030 to YZ and grant number 81700550 to GS.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.


Salmonella enterica str. SL1344 influences global supercoiling and results in an altered transcriptome with increased expression of stress response pathways [2]. Aminocoumarin antibiotics, such as novobiocin, can be used to relax supercoiling and alter the expression of supercoiling-sensitive genes. Meanwhile, Salmonella enterica demonstrates a significant resistance to this antibiotic and relatively small variability of supercoiling in response to the growth phase, osmotic pressure, and novobiocin treatment. Here we present for the first time transcriptome data of Salmonella enterica subsp. Enterica serovar Typhimurium str. 14028S grown in the presence of novobiocin. These data will help identify genes involved in novobiocin resistance and adaptation processes associated with torsion perturbations in S. enterica. Cleaned FASTQ files for the RNA-seq libraries are deposited in the NCBI Sequence Read Archive  and have been assigned BioProject accession PRJNA599397.In enteric bacteria, DNA supercoiling is highly responsive to environmental conditions. Host specific features of environment serve as cues for the expression of genes required for colonization of host niches via changing supercoiling [1]. It has been shown that substitution at position 87 of GyrA of  The data sets were named based on novobiocin concentration and growth time after addition of novobiocin. Information on the treatment (sampling) time and growth rate of the cultures is presented in The dataset of this article provides information on raw RNA-seq reads obtained from samples of 22.1Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028S (NCBI:txid588858). This strain was cultured on a Luria-Bertani (LB) agar plate (Sigma Aldrich) and a single colony was used to inoculate 10 mL of fresh LB broth for aerobic incubation at 37 °C. After 12 h, 1 mL of this preculture was added to 50 mL of LB broth and incubated aerobically at 37 °C until the middle of the exponential growth phase (A600 = 0.6), which was reached approximately 4 h after the initial inoculation. Novobiocin (Sigma Aldrich) was added at various concentrations directly to the flask and turbidity was monitored by measuring the optical density.Strain used in this work was 2.2Salmonella growth, but by the end of time sampling the growth of bacteria in both cases resumed. At time zero and after 60 min 10 mL aliquots were taken and bacteria were harvested and fixed. The control sample and the sample supplemented with 500 μg of novobiocin per mL were also fixed after 10 and 20 minutes. Total RNA was isolated and cDNA libraries were prepared for RNA-sequencing. Directional libraries were sequenced on Illumina Hiseq2500 in single reads. The RNA-seq raw reads were further analyzed to get the clean reads and stored in FASTQ files.The bacteria were grown in LB medium until middle log-phase where 0, 100 and 500 μg of novobiocin per mL was added. This increase in the antibiotic concentration has led to a gradual inhibition of the 2.3Bacterial cultures were fixed with an equal volume of cold RNA-stabilizing solution  on ice for 30 minutes. Cells were harvested by centrifugation and total RNA was extracted using RNA Extract Reagent  according to the manufacturer's protocol. DNA contaminants were removed using RNase-free DNase I kit . The integrity of the RNA was checked by Agilent 2100 bioanalyzer (USA). rRNA depletion performed using Ribo-Zero rRNA Removal Kit for Gram-Negative Bacteria . Barcoded RNA libraries were generated. NEBNext Ultra Directional RNA Library Prep Kit for Illumina was used to prepare RNA-seq libraries. The resulting average size of the cDNA libraries was approximately 300 bp. Libraries were sequenced using the Illumina HiSeq 2500 sequencing platform.2.4http://jgi.doe.gov/data-and-tools/bb-tools/) to remove Illumina adapters, NEB indexes and to eliminate rRNA reads (ktrim = r k = 23 mink = 11 hdist = 1 tpe tbo minlen = 25).266,853,687 reads were obtained in total with a length of 60 nucleotides. Raw Fastq files and clean reads were quality controlled using FastQC software (Version 0.11.5) . Raw rea2.5Salmonella enterica subsp. enterica serovar Typhimurium strain 14028S assembly GCA_000022165.1 .Filtered high-quality reads were mapped onto the genome sequence of the HISAT2 version 2.1.0  was used