Recently, it has been suggested that RNA editing may be more frequent than previously thought. A great impediment, however, to a deeper understanding of this process is the paramount sequencing effort that needs to be undertaken to identify RNA editing events. Here, we describe an in silico approach, based on machine learning, that ameliorates this problem. Using 41 nucleotide long DNA sequences, we show that novel A-to-I RNA editing events can be predicted from known A-to-I RNA editing events intra- and interspecies. The validity of the proposed method was verified in an independent experimental dataset. Using our approach, 203 202 putative A-to-I RNA editing events were predicted in the whole human genome. Out of these, 9% were previously reported. The remaining sites require further validation, e.g., by targeted deep sequencing. In conclusion, the approach described here is a useful tool to identify potential A-to-I RNA editing events without the requirement of extensive RNA sequencing.RNA editing is a post-transcriptional alteration of RNA sequences that,  RNA editing is a post-transcriptional processing of the RNA molecule, involving insertions and deletions of bases, as well as base substitutions. RNA editing has been observed in eukaryotes as well as viruses, archaea and prokaryotes, and has attracted broad interest recently –4. UntilAccurate identification of RNA editing events from high throughput sequencing data still remains a great challenge. Current comparative methods usually map short reads to a reference genome or transcriptome, followed by removal of identical reads, filtering of low quality reads, calling variants and discarding of known single nucleotide polymorphisms (SNPs) , 10–14. To overcome some of these problems, we here introduce a machine learning approach  to detecThe Darned database Sep 2013), used in this study, was created by Kiran and Baranov  [20]. It13, used CD-HIT  , specificity (Sp), accuracy (Acc), positive predictive value (PPV), Matthew's Correlation Coefficient (MCC) and geometric mean (G mean) , were usA-to-I RNA editing events were called from RNA sequencing (RNA-seq) data generated from human islets from 184 donors (Data partly published and deposited in the Gene Expression Omnibus (GEO) database, accession no. GSE50244). Details of RNA-seq and exome sequencing (Exome-seq) experiments can be found in a recent study . 101-bp http://figshare.com/projects/Discriminative_prediction_of_A-to-I_RNA_editing_events_from_DNA_sequence/15510/. Training and testing datasets are also deposited within this link.The source for one-class and binary classifications is modified based on LIBSVM. It is available at First, we made a descriptive analysis using the Darned database  Fig 2)..Fig 2). Subsequently, 10 nucleotides (nt) long flanking sequences surrounding the coordinate of the A-to-I RNA editing event (editing-sequence contexts) were extracted from the reference genome . A comparison of similarity among the three species by CD-HIT , a progrNext, we investigated the redundancy of A-to-I editing-sequence contexts in each species by using CD-HIT with a cut off of 0.8 see . For theTo find the optimal length of editing-sequence contexts for prediction, we employed the fruit fly as model. We extracted 5nt, 10nt, 15nt, 20nt and 25nt long flanking sequences surrounding the A-to-I RNA editing events  from the fruit fly genome (dm3). Considering that positive instances and negative instances in the fruit fly data were imbalanced, we oversampled the positive data set 2.31-fold. As displayed in A 5-fold cross validation was performed to evaluate which weight w to use for fruit fly, mouse and human data. An oversampling approach was used to balance positive and negative instances for mouse (1.92-fold oversampling for the positive data set). The positive human data set was assembled from the representative sequences of clusters see . The majA receiver operating characteristic (ROC) curve was plotted for fruit fly (w = 0.5), mouse (w = 0.2) and human (w = 0.2). The AUC (area under the curve) values for fruit fly, mouse and human were 0.756, 0.857 and 0.791, respectively. In line with results displayed in Using results of five-fold cross-validation for drawing ROC curve when weight w = 0.2, we evaluated the performance of the binary classifier separately on regions of Alu elements and non-Alu elements of human A-to-I RNA editing events, respectively. 6544 out of 7375 events (88.7%) in Alu regions and 12500 out of 18853 events (66.3%) in non-Alu regions were correctly recognized, respectively .Not surprisingly, one-class classification of the positive data sets, using a default nu value see  of 0.5, Next, we employed one-class classification models built on fruit fly and mouse data to study A-to-I RNA editing events in humans . With a Due to the differences between models trained on the different species, we trained a model on combined fruit fly and mouse data. With this mixed-model, prediction of editing events in human data was improved with respect to sensitivity . Remarkably, the PPV values were above 0.700 in all three animal models, indicating a low false discovery rate. Hence, models generated on fruit fly and mouse data reliably recognized human editing events.To assess the predictive capability of our models, we culled human A-to-I editing events from a Sanger sequencing data set  . Out of One-class classification  recognizOur approach outperformed an existing predictor InosinePredict , whose aFirst, we took 26 228 representative editing-sequence contexts from clusters obtained by CD-Hit see  as seedsThe genomic distribution of these putative RNA editing sites is shown in There were 561 putative A-to-I RNA editing events in exons out of the reported putative events left after removing overlap with DARNED and RADAR. First, we validated these sites using RNA sequencing of human islets from 184 donors . 322 werIn contrast, we also investigated all (3893) known exonic A-to-I RNA editing events in the human training set. After filtering 902 low quality sites, 122 no-call sites and 506 SNPs, 1352 out of 2363 (57.2%) known editing sites were identified by RNA-seq in the human islets alone . Of thesAfter this initial validation using RNA-seq alone, we further validated these novel exonic A-to-I RNA editing events, using novel Exome-seq and RNA-seq data from 12 recently analyzed human islet donors. We identifed 47 editing sites by RNA-seq, out of which 35 could be validated comparing genotypes derived from Exome-seq with that of RNA-seq. However, genotypes of 11 sites could not be inferred from Exome-seq, and were thus potential artefacts from sex chromosomes. Only one site failed to be validated. Interestingly, 4 SNPs were also found to be sites of A-to-I RNA editing events in human islets .Identification of RNA editing events is of great importance for the understanding of post-transcriptional regulation. Currently, most editing sites have been identified by large scale sequencing efforts. However, these studies are expensive and time-consuming. In addition, it is difficult to distinguish editing events from SNPs and sequencing errors. Furthermore, whereas we today have access to huge amounts of genomic data, we rarely have access to both DNA and RNA sequences from the same individual. Currently, a targeted analysis of RNA editing remains a challenge, since information on where they occur is lacking. A recent study of ultra-deep Alu-targeted sequencing showed that the scope of editing is much greater than expected . Most idTo avoid overfitting, we performed predictions based on models derived from one species on data from other species in addition to validation, using Sanger sequencing-verified RNA editing events. To this end, models trained on human data or combined data from fruit fly and mouse correctly recognized more than 70% of verified RNA editing sites. The successful identification on this independent data set validated the proposed method. From the study on currently known RNA editing events, we concluded that A-to-I RNA editing events could be predicted by use of the DNA sequence. However, we also believe that use of more data is likely to generate a higher accuracy of prediction.Considering that the negative data sets that we used for binary classification may contain novel editing events, we used a one-class classification approach to overcome this problem. With this approach, we could predict A-to-I RNA editing events using only a positive data set. However, the accuracy of this approach was slightly lower than for binary classification. This was likely due to that most editing events were merged into a single cluster. The non-editing events might be mistaken as editing events if they were located in the distribution of this one cluster. One solution to this problem would be to divide A-to-I RNA editing events into numbers of clusters and to perform one-class classification on each cluster. Another possible solution to address false positive is enlarging negative data sets used in the binary classification. However, this approach would result in extremely low sensitivity and MCC.String distances have for long been applied to measure similarity of DNA or protein sequences . We founBy scanning the whole human genome, it was surprising to find a large number of 41nt-length sequence segments, using 26 228 human editing-sequence contexts as seeds. With a maximum of 3 nucleotide mismatches and no indel, we found 25 487 374 matched segments, revealing that RNA editing may be very frequent in humans. Considering that this number largely exceeds the number of currently known A-to-I RNA editing events and that there might be tissue-specific editing events, it is likely that currently unknown mechanisms control RNA editing. A prediction using only exactly matched sequences yielded 203 202 potential A-to-I RNA editing events. Among those, most were located in noncoding regions. At this point, the role of RNA editing in these regions remains unclear. We also predicted several thousands A-to-I RNA editing events in long non-coding RNA (lncRNA). Further investigations on these specific RNA editing events would be attractive as lncRNA plays an important role in cellular regulation, such as pre-mRNA processing and splicing, transport, translation, and degradation.As previously reported, A-to-I RNA editing events were rarely found in human pancreatic islets. Nevertheless, we were able to validate 35 A-to-I RNA editing events in this tissue, based on newly analyzed Exome- and RNA- seq data from 12 batches of human islets not included in a previous report from our centre . It is cIn conclusion, we have shown that A-to-I RNA editing events can be predicted by the flanking genome sequences. The very large number of putative editing sites identified in the whole human genome may become a resource, which can be further investigated and validated using targeted deep sequencing or PCR.S1 Dataset(XLS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


Haloferax mediterranei has been used as a model microorganism. The bioinformatic approach has allowed for the prediction of 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-Sequencing (RNA-Seq). The secondary structure of these sRNAs and their possible targets have been identified. Curiously, some of them present as possible target genes relating to nitrogen assimilation, such as glutamate dehydrogenase and the nitrogen regulatory PII protein. Analysis of RNA-Seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and bioinformatic approaches used in this work have allowed for the identification of new sRNAs in H. mediterranei, some of which show different expression patterns depending on the nitrogen source. This suggests that these sRNAs could be involved in the regulation of nitrogen assimilation and can constitute an important gene regulatory network.Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in the domain Archaea and their possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea,  The complementarity between the sRNA and its target sequence is not complete and, for this reason, they require the presence of RNA chaperones to facilitate nucleotide binding [cis-encoded sRNAs originate in the nonsense strand of an open reading frame (ORF). Generally, that ORF corresponds to the sRNA target which presents complete complementarity to the cis-encoded sRNA. In addition, there have also been other types of RNA identified in Archaea: small nucleolar RNAs (snoRNAs) involved in the modifications of ribosomal RNA, whose presence was originally believed to be restricted to eukaryotic organisms [Small RNAs (sRNAs) play an essential role in the post-transcriptional regulation of many cellular processes in all domains of life, i.e., Eukarya, Bacteria, and Archaea. In eukaryotes, there are different classes of these, the best studied being microRNA (miRNAs), small interference RNA (siRNAs), and piwi-interacting RNAs (piRNAs). These are approximately 20–30 nucleotides (nt) in length and are involved in development, cellular activities, and different physiology processes ,2,3. In ed sRNAs . Trans-e binding . In contrganisms ; sRNAs irganisms ; and sRNrganisms .Archaeoglobus fulgidus [Sulfolobus solfataricus [Pyrococcus abyssi [Methanosarcina mazei [Haloferax volcanii [Pyrobaculum sp. [Thermococcus kodakaraensis [In Eukarya and Bacteria, many sRNAs have been characterised in detail. However, in domain Archaea, though there has been considerable progress in recent years, the number of sRNAs characterised is considerably lower than those in the other two domains ,13,14. Tfulgidus , Sulfoloataricus , Pyrococs abyssi , Methanona mazei , Haloferulum sp. , and Thearaensis . In thesaraensis . This faHaloferax mediterranei is an extremely halophilic archaeon which belongs to the lineage of Euryarchaeota. This microorganism grows optimally at 2.5 M NaCl [H. mediterranei as a haloarchaeal model. Specifically, biochemical, physiological, and transcriptomic studies of the assimilatory pathway in the presence of different nitrogen sources have been previously performed [H. mediterranei [5 M NaCl  in a def5 M NaCl ,22. Mosterformed ,24,25, aterranei . These rH. mediterranei expressed under two different nitrogen sources, nitrate and ammonium, using a combination of RNA-Seq and bioinformatic approaches. The main aim of this work is to extend the understanding of the global regulation network of the assimilatory pathway in H. mediterranei, specifically, and in domain Archaea, generally.In this work, we present an analysis of putative sRNAs in H. mediterranei strain R4 (ATCC 33500T) was grown at 42 °C with aeration at 225 rpm, contained in a 25% (w/v) mixture of inorganic salts  [H. mediterranei was grown in two different nitrogen sources, in a defined medium which contained 40 mM KNO3 or 40mM NH4Cl and supplemented with 5 g/L glucose, 0.0005 g/L FeCl3, and 0.5 g/L KH2PO4. Three independent biological replicates of each condition were employed.t water) . The pH H. volcanii [M. mazei [S. solfataricus [A. fulgidus [H. mediterranei strain R4  using BLASTn [E-value < 0.05, p-value < 0.05) were prioritised using a function of identity, lower number of gaps (maximum 3), and lower number of mismatches.The library of candidate sRNAs was constructed from putative sRNAs obtained in four different species of Archaea: volcanii , M. mazeM. mazei , S. solfataricus , and A. fulgidus . All putg BLASTn . The seqH. mediterranei was grown in the presence of two different nitrogen sources, nitrate and ammonium, to mid-exponential phase. The mid-exponential growth phase was reached at different times and values of OD600 nm depending on the nitrogen source [mirVanaTM miRNA isolation kit  following product specifications. Afterwards, the RNA samples were treated with Turbo DNase . RNA concentration was analysed by means of a Nanodrop ND-100 Spectrophotometer , and the quality was analysed using the Small RNA Analysis Kit on Agilent 2200 Tapestation , respectively.For RNA isolation, n source . RNA wasLibrary preparation and sequencing were performed by the Bioarray, S.L. company . sRNA libraries were constructed using TruSeq Small RNA Library Prep  and sequenced on an llumina HiSeq 2500 system using a 50-base pair (bp) read length. The corresponding FASTQ files were obtained as a result.H. mediterranei ATCC 33500 genome  as a reference. Seven ammonium BAM files and eight nitrogen BAM files were viewed in the Integrative Genomics Viewer (IGV) program [H. mediterranei genome.Raw reads from each FASTQ file were aligned into BAM files—compressed binary file used to represent aligned sequences up to 128Mb  for each program ,32, alonH. mediterranei genome, were analysed manually using the Integrative Genomics Viewer (IGV). Only sequences with a score of at least 20 (frequency of reads in high-throughput sequencing) and that were present in the six samples of each condition were selected as sRNAs in H. mediterranei. The candidate sRNAs were classified as cis-encoded sRNA if they were encoded in the reverse direction of ORF, as trans-encoded sRNA if they were in intergenic regions, and as crRNA if they were in a CRISPR array. Since the size of the readings was generally greater than the homology shown in the different species analysed, the BAM files of each condition were pooled, thus, increasing the coverage of the readings, allowing more precise limiting of the sRNAs location in the H. mediterranei genome. Once the positions were obtained, the approximate sizes of the sRNAs were calculated. In addition, the genetic environment of each sRNA was analysed manually. The raw readings, which aligned at the positions of the library of candidate sRNAs obtained from the different species against the H. mediterranei were obtained using the intersection nonempty feature from the HT-Seq program [i in the read, a set S(i) is defined as the set of all features overlapping the i position. Then, if S contains precisely one feature, the read is counted for this feature. If S is empty, the read is counted as no feature. Finally, if S contains more than one feature, the read is considered as ambiguous and is not counted for any features.The counts of the sRNAs of  program . In thisp-value < 0.01 and p-adj < 0.05). Through this method, the variance-mean dependence in count data was estimated and tested for differential expression; this was based on a model using the negative binomial distribution between nitrate and ammonium group samples.Differential expression analysis in the function of the nitrogen source (nitrate/ammonium) was performed using the DESeq2 library from the Bioconductor 3.5 package  obtaininBoth raw (FASTAQ files) and processed data  are available on the Gene Expression Omnibus (GEO) database (Series entry number: GSE108616) .cis-encoded sRNAs antisense of characterised ORF were analysed using IntaRNA [Mfold was used to predict the secondary structure of sRNAs obtained . Potenti IntaRNA . BLASTn  IntaRNA  was usedTM Low Range DNA Ladder  run in parallel. PCR products were purified with the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit  and confirmed by Sanger sequencing .The validation of 20 sRNAs was performed by reverse transcription polymerase chain reaction (RT-PCR), prioritising those with differential expression based on the nitrogen source and/or target genes of known functions. The RNA was isolated as described before. Between 0.5 and 0.8 µg of DNA-free RNA was used for the synthesis of cDNA using M-MuLV Reverse Transcriptase  and random hexamer primer  according to the manufacturer’s instructions. Negative controls for reverse transcription polymerase chain reaction (RT-PCR) were prepared by omitting reverse transcriptase and cDNA. The oligonucleotides used to carry out the RT-PCR were designed based on the sequence of sRNA candidates . AmplifiH. volcanii [M. mazei [S. solfataricus [A. fulgidus [H. volcanii aligned at many positions in the genome of H. mediterranei , the results obtained were prioritised by a function of identity, lower number of gaps (maximum 3), and lower number of mismatches. Given the evolutionary distance, the candidate sRNAs of the remaining species aligned in fewer positions  than H. volcanii in the genome of H. mediterranei, so it was not necessary to prioritise in the same way. From the bioinformatic analysis, a library of 295 candidate sRNAs in H. mediterranei were obtained  being intergenic (trans-encoded sRNA).The results of the localisation and classification of the sRNAs verified by RNA-Seq reveals that 58 sRNAs of the 116 sRNAs predicted by homology with volcanii  could beed sRNAs , of whicataricus , only eifulgidus , only siUsually, sRNAs are characterised by stable secondary structures. In order to analyse the secondary structure of the 88 putative sRNAs, Mfold software  was usedcis-encoded sRNAs antisense of characterised ORF were analysed using IntaRNA [cis-encoded sRNAs analysed present high interaction energy and full complementarity with the ORF where they were identified. The combination of these software programs has allowed the identification of possible gene targets for more than 70% of the 88 possible sRNAs identified by RNA-Seq. However, there are still 26 sRNAs whose possible targets could not be found with the software employed.The identification of potential sRNA targets was carried out via a bioinformatic approach using TargetRNA2 . Gene ta IntaRNA  . Only hits with a nucleotide identity higher than 60% combined with a coverage between the query and subject sequence higher than 80% were considered as conserved. The results of homology are shown in The sequence conservation of the sRNAs predicted in g BLASTn , compariH. mediterranei according to the nitrogen source was performed using the HT-Seq program, which counts the readings of the sRNAs verified by RNA-Seq and DESeq2, which carries out the differential expression analysis (nitrate/ammonium). From the 88 candidate sRNAs analysed, 16 sRNAs met the statistical criteria with a p-value and p-adj lower than 0.02 and 0.05, respectively (2-fold-change between 0.519 and 8.699), whereas the other eight present a decrease in their transcriptional level in presence of ammonium (log2-fold-change between −0.478 and 1.325).The expression analysis of the sRNAs in ectively . These sTo confirm the presence of 20 sRNAs predicted by RNA-Seq we used RT-PCR ,42,43,44The majority of sRNAs  are expressed in both conditions, in the presence of nitrate and ammonium. However, three sRNAs show a different expression pattern according to the nitrogen source; HM8_S and HM1_M sRNAs are expressed exclusively in the presence of nitrate and HM16_M sRNA is expressed in the presence of ammonium.H. mediterranei. Most of them are located on the chromosome, as in the case of H. volcanii. However, there is a difference between these two halophilic microorganisms. H. mediterranei sRNAs are predominantly intergenic (trans-encoded sRNA), whereas H. volcanii sRNAs are mostly antisense instead [Two different approaches, bioinformatic and RNomic, have allowed the identification of 88 sRNAs in  instead ,45. H. mediterranei were predicted using TargetRNA2 [H. mediterranei is closely related to the nitrogen source [H. mediterranei contains one gene encoding the Lsm1 protein (HFX_2733) in its genome that shares 99% sequence identity with the H. volcanii RNA-binding Lsm protein . These results suggest that the interaction between Lsm proteins and sRNAs and their participation in the RNA metabolism are also possible in H. mediterranei. Transporters are also included in the different targets predicted, specifically metal and ABC transporters, which have been observed in other analyses of bacterial sRNAs such as Paracoccus denitrificans [Ruegeria pomeroyi [S. solfataricus [H. volcanii [S. solfataricus allowed for the identification of one sRNA in that microorganism which suggests a direct interaction of these proteins with sRNAs [cis-encoded sRNAs antisense of ORF characterised in H. mediterranei were also analysed using IntaRNA [cis-encoded sRNAs. Four of them were present in only one target and the other five could interact with other possible targets. Most sRNAs that did not exhibit interaction with RNA using IntaRNA did exhibit interaction with other mRNAs using TargetRNA2. Therefore, it is likely that some sRNAs are specific to a gene, while others have a broader range of action, being involved in several metabolic pathways . Of these, eight show overexpression when H. mediterranei grows with nitrate as the sole nitrogen source, whereas the other eight show a decrease in their transcriptional level in the presence of ammonium. The majority of sRNAs validated by RT-PCR are expressed in the presence of nitrogen sources nitrate and ammonium. However, HM8_S and HM16_M sRNAs are expressed exclusively in the presence of nitrate and ammonium, respectively. Curiously, although the HM1_M sRNA transcript level does not show differences in the RNA-Seq analysis according to the nitrogen source, RT-PCR results clearly reveal that this sRNA is only expressed in the presence of nitrate (The transcript pattern analysis of  nitrate . amt1 gene (H. mediterranei have confirmed that ammonium transporters are expressed in the presence of nitrate or under nitrogen starvation [Interestingly, some of the sRNAs which show differences in their expression pattern according to the nitrogen source possibly have target genes whose expression also depends on the nitrogen source. HM8_S, which is overexpressed in nitrate as the nitrogen source, has the protein glutamate dehydrogenase as a possible target . Glutamamt1 gene , which earvation . Hence, H. mediterranei using bioinformatic and RNomic approaches, some of which show different expression patterns depending on the nitrogen source and/or present genes involved in the nitrogen assimilation as a potential gene target. This data suggests that some of these sRNAs could be related to the regulation of nitrogen assimilation, being able to constitute an important gene regulatory network which involves enzymes, transporters, and transcriptional regulators in this metabolism. Undoubtedly, this work constitutes an excellent starting point to elucidate the role of these sRNAs in the nitrogen metabolism of haloarchaea. This work, focused on identifying sRNAs involved in nitrogen assimilation, has also increased the knowledge about sRNAs in the domain Archaea. Specifically, 88 sRNAs have been identified in


CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.The online version of this article (doi:10.1186/s13059-017-1381-1) contains supplementary material, which is available to authorized users. CRISPR/Cas adaptive immunity systems confer resistance to invading phages and conjugative plasmids in bacteria and archaea –3. CRISPUsing microbial genome data mining and computational prediction approaches to search for previously unexplored class II CRISPR Cas systems has led to the discovery of novel Class II CRISPR systems, including C2c1, C2c2, and C2c3 . C2c1 anLeptotrichia shahii (LshCas13a) has shown that the single effector Cas13a protein is indeed a programmable RNA-guided single-stranded RNA (ssRNA) ribonuclease [Escherichia coli by interfering with the MS2 lytic ssRNA phage. Cas13a guided by a crRNA containing a 28-nt spacer sequence cleaves target ssRNAs in vitro and in vivo with protospacer flanking sequence (PFS) of A, U, or C. Cas13a catalytic activity is affected by the secondary structure of the target sequence, where Cas13a tends to preferentially cleave at uracil residues of multiple sites within secondary structures formed in the ssRNA targets. Moreover, the two HEPN domains are responsible for the RNA cleavage activity of Cas13a; mutation of putative catalytic arginine residues within both domains abolished the RNA cleavage activity, resulting in a catalytically inactive version of the Cas13a enzyme (dCas13a). However, like dCas9, dCas13a retains its ability to bind specifically to the target RNA and therefore can function as an RNA-guided RNA-binding protein [E. coli. However, E. coli cells expressing RFP and active CRISPR/Cas13a machinery targeting RFP transcript exhibited reduced growth, indicating collateral RNA degradation activity. The collateral Cas13a RNA degradation activities require the successful targeting of the target sequence and the activation of the Cas13a protein. Such collateral targeting could provide a means for engineering programmed cell death for biotechnological applications [A recent work characterizing the functionality of Cas13a of nuclease . Cas13a  protein , 14. Casications .Leptotrichia buccalis is activated by a specific crRNA to target a specific ssRNA sequence, but this activation led also to collateral non-specific activities, leading to cell toxicity [East-Seletsky et al.  have shotoxicity . The stutoxicity . Findingtoxicity .R genes, into crop plants has gained prominence as an effective approach to engineer transgenic plants resistant to virus infection [Plant RNA viruses are responsible for a significant proportion of commercially important plant diseases, infecting a wide range of plant species and resulting in severe losses in quality and quantity in diverse key crops , 19. Varnfection –27. Despnfection –30.Potyviruses are plant-infecting viruses that belong to the Potyvirus genus, one of the largest groups of plant viruses besides the Begomoviruses. Potyviruses represent an economically important group of pathogens capable of infecting and causing a serious damage to a wide range of crop plants. Members of the Potyviruses, such as Turnip mosaic virus (TuMV), possess long, filamentous particles that are 700–750 nm long, harboring a positive-stranded RNA genome of about 10,000 nucleotides. The ssRNA genome encodes a single long open reading frame (ORF) flanked by terminal untranslated regions. The RNA genome is translated into a single large polyprotein, which is subsequently cleaved by virus-encoded proteinases to yield at least ten functional proteins [proteins .in planta applications. Our study reveals that CRISPR/Cas13a catalytic activities resulted in interference against TuMV-GFP in transient assays and stable overexpression lines of Nicotiana benthamiana and that Cas13a can process long pre-crRNA transcripts into functional crRNAs, resulting in TuMV interference. Thus, our study demonstrates that the CRISPR/Cas13a system is portable to plant species for RNA virus interference, thereby opening myriad possibilities for research and applied uses in plants and other eukaryotic species.Here, we sought to investigate the possibility of adopting the CRISPR/Cas13a system for interference against RNA viruses in plants and, to that end, we engineered the CRISPR/Cas13a RNA interference system for Turnip mosaic virus (TuMV), of the family Potyviridae, as a target for CRISPR/Cas13a. A recombinant TuMV (TuMV-GFP) expressing GFP was used to provide an easy-to-monitor system for virus infection, replication, and spread, thereby facilitating the assessment of the interference activity of CRISPR/Cas13a system against the RNA virus.Because CRISPR/Cas13a defends bacterial cells against invading RNA viruses, we attempted to test the feasibility and portability of the CRISPR/Cas13a system to provide molecular immunity against RNA viruses in plants. To this end, we selected the potyvirus Leptotrichia shahii Cas13a  nucleotide sequence for expression in plants and custom synthesized four overlapping fragments to assemble the construct, which we dubbed plant-codon optimized Cas13a (pCas13a). The four overlapping fragments of pCas13a were assembled by unique restriction enzymes to generate a full-length clone flanked by attL1 and attL2 recombination sites, a nuclear localization signal fused to the C-terminus of the protein, and a 3x-HA tag fusion at the N-terminus to facilitate protein detection. Next, pCas13a was sub-cloned into the pK2GW7 destination binary vector via Gateway recombination reaction to generate pK2GW7:pCas13a over-expression clones driven by the Cauliflower mosaic virus (CaMV) 35S promoter . We confirmed the pCas13a expression in transient assays in N. benthamina leaves and in permanent lines over-expressing pCas13a. Our western blotting results with anti-HA tag antibody demonstrated that the pCas13a clone was expressed in plants and the correct protein size was detected , and the helper component proteinase silencing suppressor (HC-Pro) and coat protein (CP) sequences  to transiently and systemically express crRNAs under the Pea early browning virus (PEBV) promoter, as previously described , as well as TuMV-GFP infectious clones co-delivered into N. benthamiana leaves via agro-infection. In addition, a non-specific crRNA (ns-crRNA) with no sequence similarity to the TuMV-GFP genome was used as a control. The interference activity of the CRISPR/pCas13a system against the TuMV-GFP virus was assessed at seven days post infiltration (dpi) by visualizing the GFP signal in the plant systemic leaves under ultraviolet (UV) light. We observed a reduction of ~ 50% in the level of GFP signal in the systemic leaves of plants with crRNAs targeting the HC-Pro and GFP2 sequences. In addition, a low, but detectable reduction in the GFP signal was observed with the CP and GFP1 crRNAs compared to the control, whereas no difference in the GFP signal was observed in the wild-type (WT) N. benthamiana plants co-infiltrated with the TRV expressing ns-crRNAs, targeted crRNAs, and TuMV-GFP but not pK2GW7:pCas13a clones  under the control of CaMV35S promoter. These transgenic plants were generated via Agrobacterium-mediated transformation of pK2GW7:pCas13a clones into WT N. benthamiana plants; and the pCas13a protein expression was confirmed by western blotting   Fig. .Fig. 3pCConsidering the possibility that the processed “mature” crRNAs would assemble with pCas13a to form active pCas13a–crRNA complexes, we investigated whether the generated mature crRNAs are functional and therefore would allow efficient targeting of the TuMV-GFP RNA genome. The pCas13-OE plants were agro-infiltrated with TRV expressing either the poly-crRNA or the ns-poly-crRNAs and were challenged with the TuMV-GFP virus. The viral GFP signal was determined in systemic leaves at 7 dpi. Strikingly, we detected a significant reduction in the intensity and spread of the viral GFP signal in plants with poly-crRNA. On the other hand, no reduction in the GFP signal was observed with the ns-poly-crRNA relative to the control Fig. . A quantCRISPR/Cas systems provide bacteria and archaea with immunity to fend off invading nucleic acids; and thus provide synthetic bioengineers with rich resources to build systems to edit and regulate the genome and epigenome; and to modulate, modify, and monitor the transcriptome. Therefore, CRISPR/Cas systems can be harnessed for functional biology, biotechnology, and genetic medicine. Here, we harnessed CRISPR/Cas13a to selectively interfere with the TuMV RNA virus. Our data demonstrate that CRISPR/Cas13a can mediate molecular interference against a plant RNA virus. Targeting different viral genomic regions for degradation resulted in reduced levels of GFP, coupled with the reduction in the accumulation of the TuMV-GFP RNA genome in systemic leaves. These findings indicate the attenuation of the TuMV virus replication and spread, corroborating the effectiveness of CRISPR/pCas13a in targeting and interfering with the TuMV RNA viruses in plants.HC-Pro and GFP2 sequences. However, crRNAs targeting the CP or GFP1 target sequences were less effective, suggesting that, among other unexplored factors, secondary structures within the target TuMV-GFP RNA or the presence of RNA binding proteins might influence the accessibility and thus the activity of the pCas13a on the target RNA. This observation is consistent with other studies that have shown that the activity of Cas13 is highly governed by RNA target accessibility. For example, Abudayyeh et al. have characterized the LshCas13a activity in its native environment and have shown that the most effective crRNAs were found to be clustered and closer together in regions of strong interference, suggesting that target RNA accessibility, indeed, impacts the Cas13a activity [In this study, we observed that virus interference was most efficient with crRNAs targeting sequences within the activity . In addiactivity  and BzCaactivity  CRISPR sactivity .N. benthamiana plants. Our results . Both the poly-crRNA and the ns-poly-crRNAs were cloned to the TRV RNA2 by BamHI and SacI.N. benthamiana plants expressing Cas13a were regenerated using the previously developed protocol [pK2GW7-pCas13a construct was used to generate plants (N. benthamiana or resistant) for programming resistance to TuMV , and incubated at ambient temperature for 2 h. For infiltration into Cas13a-OE plants, cultures were mixed at an OD600 ratio of 0.05:1:1  and infiltrated into three- to four-week-old leaves of N. benthamiana Cas13a-OE plants with a 1-mL needleless syringe. GFP expression was observed at 3, 7, and 10 dpi using a hand-held UV light. Photos were taken with a Nikon camera both in normal light and under UV light. GFP signal quantifications were done using ImageJ software. Leaves samples were collected at 7 and 15 dpi for molecular analyses.Constructs, harboring the TuMV-GFP infectious clone, TRV RNA2 empty, or containing crRNA under PEBV promoter, TRV RNA1, were individually electroporated into 2VO3, NaF, MG132, and MG115. Proteins were separated on a 10% polyacrylamide gel. Immunoblot analysis was carried out using mouse α-GFP  for TuMV GFP and rat α-HA (1:500) antibody for pCas13a. The antigens were detected by chemiluminescence using an ECL-detecting reagent (Thermo Scientific).Total proteins were extracted from 100 mg of sample using extraction buffer  membrane and hybridized with a DIG-labelled probe. For virus expression analysis, a DIG-labelled RNA probe was synthesized using DIG Northern Starter Kit (Roche) and manufacturer’s instructions were followed. For crRNA detection, a 5’-end DIG-labelled oligonucleotide (IDT) was used. DIG application manual (Roche) was followed for capillary transfer, hybridization, and detection. Northern blots were repeated in three independent experiments with the same results.Total RNA was extracted from virus-infected plants using the Direct-zol RNA MiniPrep Plus (Zymo Research) according to the manufacturer’s recommendations. For each sample, 10–15 μg of RNA was separated on a denaturing 2% agarose gel, blotted on a Hybond-NAdditional file 1: Figure S1.pCas13a expression in planta. Figure S2. pCas13a-mediated interference with TuMV-GFP in planta. Figure S3. GFP quantification of TuMV-GFP interference in transgenic pCas13a-OE plants. (PDF 1393 kb)Confirmation of Additional file 2: Sequence S1:Sequence S2: pCas13a full-length plant codon optimized DNA sequence (3x-HA-pCas13a-nls) Map S1: pCas13a sequence in pK2GW7-pCas13a (pK2GW7-3xHA-pCas13a-nls). Sequence S3: TuMV-GFP full-length sequence with target sequences in different colors. Map S2: Map of TuMV-GFP. Sequence S4: Cas13a-repeat-cRNA-TuMV-GFP-GFP-target 1 sequence (A), map (B) and its complex with targeting region in TuMV GFP genomic region (C). Sequence S5: Cas13a-repeat-cRNA-TuMV-GFP-GFP-T2 sequence (A), map (B), and its complex with targeting area in TuMV GFP genomic region (C). Sequence S6: Cas13a-repeat-cRNA-TuMV-GFP-HC-Pro-T1 sequence (A), map (B), and its complex with HC-pro targeting region in TuMV (C). Sequence S7: Cas13a-repeat-cRNA-TuMV-GFP-Cp-Pro-T1 sequence (A), map (B), and its complex with Capsid protein targeting region in TuMV. (DOCX 414 kb)pCas13a amino acid sequence (3xHA-pCas13a-nls). Additional file 3: Table S1:Table S2: Primers used in this study. (DOCX 21 kb)TuMV genome description.


Escherichia coli DNA topoisomerase I has low RNA topoisomerase activity and that the R173A point mutation abolishes the unknotting activity for ssRNA, but not for ssDNA. Furthermore, we discover the topological inhibition of reverse transcription (RT) and obtain different RT–PCR patterns for an ssRNA knot and circle of the same sequence.Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. Synthetic RNA topological structures are significant for understanding the physical and biological properties pertaining to RNA topology, and these properties in turn could facilitate identifying naturally occurring topologically nontrivial RNA molecules. Here we show that topological structures containing single-stranded RNA (ssRNA) free of strong base pairing interactions can be created either by configuring RNA–DNA hybrid four-way junctions or by template-directed synthesis with a single-stranded DNA (ssDNA) topological structure. By using a constructed ssRNA knot as a highly sensitive topological probe, we find that  In vivo, complex topologies have been identified in proteins and DNA, while their existence in RNA is still unclear. Here, the authors design synthetic topological structures containing single stranded RNA, offering tools for investigating biologically relevant questions about RNA topology. Knotting and entanglement are not only common macroscopic phenomena, but also present at the molecular level, via either random statistical threading1347981213An interesting question naturally arises regarding the importance of RNA topology. Although the recent systematic screening of the Protein Data Bank indicated the absence of genuine linear knots in known RNA structures15Escherichia coli DNA Topo III . We expect our work on synthetic RNA topological structures will stimulate research in the essentially unexplored area of RNA topology and RNA topoisomerase.Here we demonstrate that synthetic RNA topologies can be accessed either by configuring the RNA–DNA hybrid four-way junction (4WJ), or by template-directed synthesis using a ssDNA topological structure. The resulting RNA topological structures are free of strong base-pairing interactions, enabling the RNA Topo activity study of The first ssRNA knot was constructed by Seeman's helix-based methodin vitro runoff transcription, which is the most convenient and economic method to produce long RNA strands, contain both 5′- and 3′-heterogeneities. Therefore, their enzymatic ligation poses a significant challenge. To solve these problems, a 5′-end hammerhead ribozyme and a 3′-end hepatitis delta virus ribozymeTo produce closed RNA topological structures, the linear RNA strands can be ligated enzymatically. However, RNA strands synthesized by TKj(+) and 14-bp-edged for the left-handed trefoil knot TKj(−). Notably, for the helix-based method, generating positive nodes would demand the formation of left-handed Z-form RNA, which requires harsher conditions The RNA–DNA hybrid 4WJs presumably have structural properties similar to that of DNA 4WJs due to the fact that the former have been utilized in the construction of various RNA–DNA hybrid nanostructures3228TKj(+) and TKj(−) were obtained in yields of 13% and 28%, respectively (TKj(+), lane 3 for TKj(−), along with the circular  and linear  references. Similar to the previous results with DNA topological structuresTKj(+) and TKj(−) have almost identical electrophoretic mobility, which is higher than that of Cj. The resistance to digestion with RNase R proves the closed structure for knots (lanes 2 and 4) and circle (lane 6), but not for the linear species (lane 8).Experimentally, three 76-nt ssRNA scaffolds were annealed with three DNA splints and either set of four DNA staples. In the resulting assembled complex, the ssRNA scaffolds are folded by the staples to form three 4WJs (configured within the tensegrity triangle) and joined end to end by the splints . Subsequectively , comparaTo further demonstrate the high-level complexity of topological structures we can achieve with RNA, we create a hybrid Borromean rings (BR) In the field of chemical topology, molecular BR is a well-known challenging target2528383940Synthetic ssDNA topological structures constructed previously, in principle, can direct the synthesis of ssRNA structures of the same topology . The int6 bulges are designed in the DNA strand of the hybrid to provide six curving points, and they connect three outer 34-bp helices (generating a 33° torsion angle) and three inner 42-bp ones (generating a −65° torsion angle) into the closed ds knot structure  images of the ds hybrid circle  to serve as the template, and three 76-nt complementary RNA strands were then annealed onto the DNA template and ligated to form a 228-nt RNA strand . Six poltructure . If the d circle  and knotd circle  reveal td circle . The higE. coli DNA Topo III as the first enzyme possessing RNA Topo activity26E. coli DNA Topo I (denoted as Topo I afterwards for clarity) for the RNA Topo assay, and in contrast to previous reportOne of the most significant discoveries resulting from synthetic RNA topology is the identification of TKj(−) (simplified as TKj afterwards), as the topological probe, which would be converted to the ssRNA circle, Cj, in the presence of an RNA Topo activity. Additionally, four RNA structures containing strong base pairings were constructed from the same 114-nt linear RNA strand, Lh, according to Seeman's helix-based methodTKh, and circle, Ch, and the dimeric Granny knot . In contrast, the most stable topoisomers of the helix-based structures are those favouring maximum base pairings and consequently are those of more complex topology (TKh and GKh respectively). Therefore, Ch, and C2h were used as the helix-based RNA Topo probes to be compared with the junction-based TKj .We used our ssRNA negative trefoil knot, prepared . Figure TKj to Cj catalysed by increasing concentrations of wild-type (WT) Topo I. Though Topo I has RNA Topo activity, this activity is relatively low and ∼28–35% of conversion was observed when the molar ratio of Topo I to RNA is 4:1 (lane 4). Based on our previous result that Topo I catalyses the fast approximately stoichiometric unknotting of ssDNA knot within 30 minTKj, Ch is a much less sensitive RNA Topo probe , and the smaller-amount final GKh (after two strand-passage events). We also find that TK2h migrates slightly faster than TKj, though both are of the same size and topology, probably due to the different sequences or the formation of transient base pairings within TK2h even during migration in the dPAGE.po probe . Only ∼3Ch and C2h (helix-based) are not as sensitive probes as TKj (junction-based) can be explained by both the Topo I binding to ssRNA and its low RNA Topo activity. Topo I binding is expected to inhibit the formation of base pairings, countering the thermodynamic driving force for the topological conversion illustrated in Ch and C2h. This problem is further exaggerated due to the low RNA Topo activity, which necessitates a higher concentration of Topo I and ultimately leads to even more severe protein binding. The low sensitivity of the helix-based probe may account for the previous failure of finding RNA Topo activity of Topo I (ref. 32P-labelled RNA as described in the recent studyThe observation that o I ref. . BesidesdTKj)TKj), the R173A mutant was investigated regarding its relaxation activities of ssDNA and ssRNA. Interestingly, we could still detect the unknotting activity of R173A mutant for the ssDNA probe  and circular (Cj) ssRNA templates (228 nt) and the reaction mixtures were analysed by dPAGE , and the rolling-circle amplification product  was generated from Cj (lanes 3 and 4). The faint top band in lane 1 and that sticking to the well in lane 3 are probably due to the strong association of the RT enzyme with the RNA–DNA complex, even after heat denaturation and during dPAGE. After treatment with RNase H, these bands disappear (lanes 2 and 4).DNA topology affects the DNA-templated processes in the living cell, such as DNA replication and transcriptionby dPAGE . As expeTKj (lane 1). However, a smear containing multiple bands was observed with Cj (lane 2) due to the tandem-repeat sequence of its cDNA. As a result, different RT–PCR patterns have been obtained for ssRNA knot and circle with the same sequence. This topology-dependent RT–PCR feature can be utilized to identify closed RNA knots from the naturally occurring circular RNAsThe cDNA products obtained were subsequently subjected to PCR with a pair of convergent primers to amplify a 167-nt region. Synthetic DNA topology plays a prominent role in the development of DNA nanotechnology462949Our current work, serving as a starting point, has greatly expanded the richness of RNA topological structures, including the first realization of RNA topoisomers of both positive and negative trefoil knots, and two different forms of RNA–DNA hybrid structures. The tools and methods that have been demonstrated or suggested in our work can help solve several unexplored problems associated with RNA topology. Aside from searching for naturally occurring RNA topological structures, whether or not there exists RNA-specific Topo is also a fundamental question worth pursuing. Though RNA Topo activity has been spotted in some proteins, there is to date no evidence supporting the cellular functions of these proteins as being directly related to the RNA Topo activity. Hypothetically, RNA Topos may correct the misfolded RNA structures or resolve RNA entanglements that could inhibit the normal functions. If so, this will open new opportunities for fundamental RNA biochemical and biophysical research, as well as for novel therapeutic inventions.in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit from the New England Biolabs (NEB). The corresponding DNA templates were generated by the PCR amplification of the gBlocks gene fragments from the Integrated DNA Technologies using the Q5 Hot Start High-Fidelity DNA Polymerase (NEB). To enhance the ribozyme cleavage, five thermal cycles were performed after transcription, with each cycle containing three steps: 70 °C for 10 s, 50 °C for 1 min and 37 °C for 10 min. The target RNA molecules were then purified by dPAGE. Each purified RNA molecule was treated with T4 polynucleotide kinase (NEB) in 1 × T4 DNA ligase buffer  at 37 °C for 6 h to remove the 2′,3′-cyclic phosphateThe design of the sequences followed the rule of sequence symmetry minimization−1), 1.5 μl of 100 mM fresh DTT (NEB) and 1.5 μl of 100 mM fresh ATP (NEB) were added and incubated at 16 °C at least 16 h for the ligation. The ds RNA–DNA hybrid structures were prepared using the corresponding ssDNA knots as templates, which were prepared according to previous workThe ssRNA topological constructs were prepared with the all-in-one protocol. It involves two steps: (1) Annealing to get the assembly complex. Equimolecular quantities  of all strands  were mixed in a buffer with the ultimate concentration of 1 × T4 DNA ligase buffer (by adding 10 × T4 DNA ligase buffer because the kinase-treated RNA scaffolds were in 1 × buffer) and annealed from 70 to 16 °C over 4 h. (2) Ligation to seal the nicks. To each 100 μl of reaction mixture, 4 μl of T4 RNA ligase 2  after the gel extraction to remove the unavoidable cleaved linear RNA during the purification. However, RNase R digestion is not useful for the hybrid BR.Gels of different concentrations were prepared using 30% acrylamide and bis-acrylamide solution  with 7 M urea in 0.5 × TBE buffer (Bio-Rad) and run on a PROTEAN II xi cell (Bio-Rad) or a Mini-PROTEAN Tetra cell (Bio-Rad). Samples were mixed 1:1 with TBE-urea sample buffer (Bio-Rad) and heated at 70 °C for 5 min before they were loaded into the wells. Gel concentrations were carefully chosen to ensure the proper separations between different topologies as well as the references. For imaging, gels were stained with GelRed (Biotium), and images were taken by Gel Doc XR+ (Bio-Rad) imaging system and processed by software Image Lab . For purification, gels (without staining) were visualized by UV shadowing against a fluorescent thin-layer chromatography plate and bands of interest were cut. The bands were then eluted using the crush-and-soak method and the eluent was purified and concentrated on 3 K Nanosep filters . The concentration of product was determined by measuring the ODVarious nucleases were used in this work, including Nt.AlwI (NEB), Nt.BspQI (NEB), RNase H (NEB), DNase I (NEB) and RNase R (Epicentre). We used the reaction conditions for these enzymes as recommended by the providers.E. coli Topo I WT and mutant proteins were expressed and purified as described in previous studyE. coli Topo I (NEB) was also tested and its RNA Topo activity was found to be slightly higher than that of the in-house prepared WT enzyme, probably due to the contamination of E. coli Topo III  and 100 μg ml−1 BSA. The concentration of substrates and proteins were described in the text. Reactions were quenched by phenol–chloroform extraction followed by ethanol precipitation. The reactions were then analysed by dPAGE.The Topo III . The rea−1 polyornithine (Sigma) solution was added to freshly cleaved mica and stand for 3 min to increase the binding to the structures before applying the samples. Then the mica was rinsed with 1 ml water and dried with compressed air. An aliquot of 5 μl of each sample (about 5 nM) in 1 × TAE-Mg buffer  was applied to the treated mica and stand for 1 min. Then the mica was rinsed with 1 ml water and dried with compressed air. AFM imaging was performed on a Veeco MultiMode 8 AFM in the ScanAsyst in air mode using the scanayst-air tips (Veeco). The AFM images were processed with the software Gwyddion.For AFM imaging of the ds RNA–DNA hybrid structures, 30 μl of 0.1 mg mlTKj or Cj) and 200 nM primer (sequence is shown in The ProtoScript II First Strand cDNA Synthesis Kit (NEB) was used for the RT. Each reaction (20 μl) contained 100 nM ssRNA template .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References.


Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples. Tendon injuries are common problems for active individuals and the aging population . TreatmeMature tendons are comprised of type I collagen, which is arranged in a highly ordered hierarchical manner along the long axis of the tissue . Tendon Previous studies have described protocols for RNA extraction from human or larger mammalian animal models such as rabbit , but anaMendias and colleagues and Nielson and colleagues have performed gene expression analysis on a single mouse Achilles or plantaris tendon in different loss-of-function mouse models or in altered loading conditions . This apThe various technical limitations associated with obtaining high-quality, high-yield RNA using existing protocols enlarges the cohorts of mice needed for statistical analysis, and can hinder the use of RT-qPCR or functional genomic assays such as RNA-seq on single adult mouse tendons. Here, we present a robust, low-cost, and straightforward RNA isolation protocol that enables the isolation of high-integrity RNA from a single mouse Achilles tendon. We show that pooling tendon samples inflates estimates of biological variance for gene expression data in RT-qPCR analysis. We apply this method to analysis of injured and contralateral uninjured tendons to demonstrate the detection of significant and reproducible gene expression changes. In addition, this method can be used to purify high quality RNA from other musculoskeletal tissues, making it easily adaptable to multiple connective and skeletal tissue types, or from difficult to obtain tissues from humans or other organisms.n = 30 total). To compare gene expression levels between injured and uninjured Achilles tendons in the same mouse, excisional Achilles tendon injuries were performed using a 0.3 mm biopsy punch as described  and high impact zirconium 1.5 mm beads . Samples were homogenized in two 180-s rounds of bead beating at 50 Hz . Samples were then moved directly to dry ice or −80 °C for longer storage up to six months.g) for 5 min, the supernatant was aspirated, and 500 μl TRIzol was added. All homogenized samples were stored at −80 °C until RNA isolation.Alternative tissue disruption procedures that were tested included homogenization of both fresh and frozen tendons in 500 μl TRIzol with a Polytron handheld homogenizer  until tissue was visibly disrupted (60–90 s). Cryogrinding of samples was tested using a freezer mill (SPEX 6875). Achilles tendons were snap frozen in liquid nitrogen and transferred to a super-cooled SPEX grinding cylinder  and pulverized in a bath of liquid nitrogen for 3 min. Ground samples were collected by rinsing the cylinder with 500 μl TRIzol and transferred to a 1.5 ml tube. For enzymatic digestion, tendons were placed in 2 ml Eppendorf tubes with 1 ml digestion solution containing 0.2% collagenase II  in DMEM  containing 0.1% Penicillin/Streptomycin  and 1% Hepes . Tubes were kept on ice during the dissection period and were incubated together in a 37 °C shaking water bath for 90 min. In order to digest remaining matrix, we added 200 μl of 0.2% collagenase I  and 300 μl of 0.4% Dispase  to the partially digested samples and incubated at 37 °C for an additional 30 min. Following the digestion, the samples were centrifuged at 500 RCF  or the Direct-Zol systems . Based on typical tendon yields, the ZymoSpin IC spin columns are optimal for use with RNA extracted from single tendons as these columns can purify up to 5 μg of RNA in as little as 6 μl eluate. However, this protocol also has been successfully used with ZymoSpin IIC columns, which require a larger elution volume. After adding the RNA/ethanol mix to the spin column, the standard Zymo purification protocol was used with the following modifications. First, a 15 min on-column DNase I treatment was added to minimize genomic DNA contamination. An extra wash step was included to improve sample purity. Prior to elution, columns were spun for an additional 2 min at maximum speed to remove residual ethanol. RNA was eluted in 15 μl RNase/DNase free water that was pre-warmed to 55–60 °C to maximize the RNA recovery from the spin column. RNA concentration was measured via fluorometric quantitation  and sample quality was determined by spectrophotometric analysis  as well as capillary electrophoresis . The final RNA product was stored at −80 °C for RT-qPCR analysis.20 primers using the SuperScript IV First Strand Synthesis System  and a no-reverse transcriptase control was included for every sample. A total of 2 ng cDNA template was amplified for 40 cycles in each SYBR green qPCR assay  using a final primer concentration of 200 nM. All assays were performed in technical triplicate using either a LightCyclerII 480  or a StepOnePlus Real Time PCR system . Three independent biological replicates were run per condition for both sets of RT-qPCR. Gapdh was used as the reference gene for all samples Scleraxis (Scx) and Gapdh technical and biological replicate cycle threshold (CT) values independently. Variance estimates for Scx ΔCT relative expression were calculated using standard error propagation techniques. Relative expression values for Collagen Ia2 (Col1a2) and SRY-Box9 (Sox9) were calculated for the injury analysis using the ΔΔCT method (n = 3 mice) were analyzed via Welch’s t-test , shearing with a handheld homogenizer , and bead beating. Capillary electrophoresis was performed on purified RNA using a Bioanalyzer RNA Nano chip . RNA integrity number (RIN), a quantification of degradation, was calculated by the accompanying Agilent software based on the electropherogram for a given sample; a RIN of ten indicates completely intact RNA whereas a RIN of one indicates severely degraded RNA. Enzymatic digestion produced intact RNA (RIN > 7), but low RNA yield (≤1 ng/μl). Cryogenic grinding and handheld homogenizer dissociation methods resulted in low yield (≤5 ng/μl) and poor RNA purity and integrity (RIN ≤ 3) . Bead beTo further evaluate our bead beating homogenization method, we performed additional experiments examining the level of degradation that occurs prior to homogenization as well as during homogenization. To address the former, single Achilles tendons from similarly aged mice were left in sterile 1× PBS on ice following dissection for up to 9 min before homogenization to simulate waiting times involved in batch dissection. The shortest time between dissection and homogenization (0–30 s) yielded more intact RNA (RIN = 6.5) while longer wait times resulted in more degraded RNA  via RT-qPCR. Although we find no gain in RNA quality from pooling, treating pools of tendons from multiple individuals as single biological replicates results in larger standard deviations in CT measurements in assays for Scx and Gapdh  and moderate (RIN = 6–7) RNA integrity, as well as efficacy in correcting for variation in RNA integrity in the differential expression analysis . AlthougOur analysis also uncovered key steps that are integral toward generating relatively intact, high yield RNA from the single tendon samples. In particular, we find that the time from dissection to homogenization and storage can significantly impact the quality of the RNA, causing measurable degradation. In this regard, even small delays on the order of minutes could affect overall RNA quality, which could greatly affect differential gene expression analysis. In addition, the duration of homogenization is important for maximizing RNA yield and quality. Homogenization times that are too short or long can result in dramatically different RIN and concentrations regardless of the level of sample pooling.Similar to previous RNA-seq studies, our RT-qPCR analysis of single and pooled tendon samples revealed that pooling increases the variance of gene expression measurements . It has Our tendon RNA extraction method is a robust protocol for obtaining high quality RNA for gene expression assays. It decreases the number of mice required for analysis and avoids extra amplification steps, making it straightforward, cost-effective, and easily accessible to researchers new to the tendon field. By providing a means for reproducibly analyzing one Achilles tendon, this method also reduces measurement error associated with pooling tendons from multiple individuals. Moreover, our protocol permits the use of internal comparisons between a limb that has undergone experimental manipulation  and the contralateral control limb within the same animal. In addition to facilitating larger-scale RT-qPCR studies, we believe this method will make highly dimensional gene expression analysis, such as RNA-seq, accessible to more researchers studying musculoskeletal tissues, thus opening new frontiers in tendon biology.10.7717/peerj.4664/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4664/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.4664/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.4664/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.4664/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.4664/supp-6Supplemental Information 6Click here for additional data file.


In recent years, with the advent of next-generation sequencing along with the development of various bioinformatics tools, RNA sequencing (RNA-Seq)-based transcriptome analysis has become much more affordable in the field of biological research. This technique has even opened up avenues to explore the transcriptome of non-model organisms for which a reference genome is not available. This has made fish health researchers march towards this technology to understand pathogenic processes and immune reactions in fish during the event of infection. Recent studies using this technology have altered and updated the previous understanding of many diseases in fish. RNA-Seq has been employed in the understanding of fish pathogens like bacteria, virus, parasites, and oomycetes. Also, it has been helpful in unraveling the immune mechanisms in fish. Additionally, RNA-Seq technology has made its way for future works, such as genetic linkage mapping, quantitative trait analysis, disease-resistant strain or broodstock selection, and the development of effective vaccines and therapies. Until now, there are no reviews that comprehensively summarize the studies which made use of RNA-Seq to explore the mechanisms of infection of pathogens and the defense strategies of fish hosts. This review aims to summarize the contemporary understanding and findings with regard to infectious pathogens and the immune system of fish that have been achieved through RNA-Seq technology. Fish endures as an important source of food and nutrition for hundreds of millions of people worldwide, and aquaculture plays a crucial role in meeting the global food demand . BecauseRNA sequencing (RNA-Seq) is an evolving modern technology that makes utilization of next-generation sequencing (NGS) or deep sequencing to deal with transcriptome profiling. Transcriptome analysis is a formidable tool that is used for a better understanding of the underlying pathways controlling cell fate, development, and disease progression in a host . Also, iIn the past two decades, the field of transcriptomics has evolved tremendously from the initial studies performed using the hybridization-based microarray technology. Microarray technology has its own limitations, such as the requirement of a prior knowledge of gene sequences from the organism of interest, the occurrence of artifacts caused by cross-hybridization, and the inability to accurately quantify low as well as high expression levels of genes ,6. AfterRNA-Seq technology, with its relatively superior advantages, has slowly replaced microarray and tag-based transcriptome analyses. The greatest advantage of RNA-Seq is that it is both qualitative and quantitative and, hence, it could measure the expression levels of even low-abundance transcripts. Furthermore, it allows the quantification of the expression levels of isoforms . Unlike RNA-Seq has already been employed in several studies to understand diseases caused by pathogens in fish and the defense mechanisms of fish against these pathogens. The transcriptome analysis during host–pathogen interaction aids in understanding the changes in the host cells during the course of infection . ParticuMajor steps involved in a transcriptome analysis using RNA-Seq are: experimental design, library construction, sequencing of the library on a NGS platform, and analysis using bioinformatic tools for interpretation . In thisThe experimental design is an important criterion for RNA-Seq-based transcriptome research. A poor experimental design may result in worthless data along with wastage of ample amounts of money and time. Hence, it is important to design the experiment with the most care and so to fulfill the research objectives. Sequencing depth, desired coverage across transcriptome, and the number of biological and technical replicates must be considered while designing the experiment. Another important thing to be considered is the type of tissue or cell population, which depends on the goals of the experiment. Conventional RNA-Seq experiments use tissue samples containing hundreds of unique cells which are heterogeneous in nature, and the results observed are confounding ,13. To oAfter the experimental design, the next step is the RNA preparation and library construction, which starts with total RNA isolation from the tissue or cell of interest. It is important to check the quality of RNA before proceeding to the further downstream step, and this can be done by using the Agilent Bioanalyzer, which provides representative values as RNA integrity numbers (RIN) lying between 1 and 10. A high RIN value represents RNA of high quality with very little degradation. Typically, RIN values lower than 6 may lead to misleading results. Total RNA is comprised of rRNA, pre-mRNA, mRNA, and various ncRNA. After the quality check, it is important to separate the particular RNA species of interest from total RNA, depending on the research objectives. The total RNA samples could be either enriched or depleted in order to obtain particular species of RNA. Poly-A selection and enrichment are done for sequencing mRNA; ribo-depletion is performed for the sequencing of mRNA, pre-mRNA, and ncRNA; size-selection strategy is followed for miRNA sequencing . The RNAThere is a rapid development and improvement of NGS technologies day by day, which fine-tune the sequencing chemistry and make the technology more affordable. Illumina, Ion Torrent, and PacBio are the most commonly available sequencing platforms in the market ,19. AmonThe bioinformatics analysis of RNA-Seq  starts wDanio rerio) is the most commonly used fish species and an excellent research model. Zebrafish embryos challenged with Salmonella typhimurium were utilized to understand the role of Traf6 function in the innate immune response [Mycobacterium marinum (M. marinum) infection [M. marinum infection in common carps (Cyprinus carpio) revealed 39 TIR domains with transcript isoforms and genes [M. marinum infections in fish are used as a model to understand tuberculosis disease in humans caused by Mycobacterium tuberculosis.Studies involving RNA-Seq technology to understand fish diseases and pathogen evasion strategies against the immune system have been performed mostly during bacterial infection . Zebrafiresponse  and tranresponse . Similarnfection . M. marind genes . M. mariEdwardsiella ictaluri  and is an economically important disease which affects the catfish aquaculture industry. Investigations on the pathogen entry mechanisms and the mucosal immune response in channel catfish  showed the importance of the actin cytoskeletal polymerization and remodeling, and of the junctional regulation of E. ictaluri entry by intestinal barrier disruption [E. ictaluri infection. A bulk segregant RNA-Seq analysis of the F2 generation of interspecific hybrids of channel catfish and blue catfish , which are respectively, relatively susceptible or extremely resistant to E. ictaluri infection, showed a higher expression of immune-related genes in the resistant fish than in the susceptible fish [Pelteobagrus fulvidraco) to E. ictaluri were established using RNA-Seq [Paralichthys olivaceus) infected with Edwardsiella tarda [Enteric septicemia of catfish is caused by sruption . Furthersruption , retinoisruption , Rab GTPsruption , and Tolsruption  in channble fish . In addi RNA-Seq . A totalla tarda .Flavobacterium columnare (F. columnare) which targets mucosal organs such as the gill and the skin, used as routes of entry. The strain of channel catfish susceptible to F. columnare infection showed a basal polarization of gill mucosal compartments possessing a putative mucosecretory and tolerogenic phenotype, leading to the evasion of the pathogen [F. columnare infection [l-rhamnose and d-galactose, against F. columnare infection. The exposure of the channel catfish to l-rhamnose and d-galactose showed a significant protection against the bacteria [F. columnare, were challenged with the same bacteria, and a comparative transcriptome analysis was done. The results suggested that in the vaccinated fish a strong expression of genes related to collagen deposition and tissue remodeling occured, compared to the unvaccinated group [RNA-Seq was used to understand the immune evasive mechanisms of pathogen . Also, anfection . It is ibacteria . In a reed group . This clAeromonas hydrophila is an opportunistic bacterial pathogen of fish found in the freshwater and brackish water environment. Different studies utilized RNA-Seq to investigate A. hydrophila infection in fishes of aquaculture importance, such as the large yellow croaker (Larimichthys crocea) [Megalobrama amblycephala) [Ctenopharyngodon idella) [Tor putitora) [Pelteobagrus vachellii) [A. hydrophila. Similarly, RNA-Seq data from a previous study was used to design a customized microarray in order to decipher the mucosal immune defense in the blue catfish during A. hydrophila infection [Oreochromis niloticus) during Streptococcus agalactiae infection revealed that toll-like receptor-mediated pathways [Oreochromis mossambicus) and enhance S. agalactiae infection [ crocea) , the blucephala) , the comcephala) , the gra idella) ,57, the utitora) , and thechellii) . These snfection . A transpathways  and miRNpathways . In addipathways . There wpathways . Also, tnfection .Cynoglossus semilaevis) [Lateolabrax japonicus) [Vibrio anguillarum identified several immune-related pathways and candidate genes during the infection progress. RNA-Seq analysis of the Japanese sea bass during Vibrio harveyi challenge revealed unique transcripts, about 8% of which were highly specific to fish and have never been reported before. Also, the study suggested that fish immune system may be more complex than previously believed, and fish might have acquired numerous fish-specific immune system components during early vertebrate evolution [Epinephelus coioides) infected with Vibrio alginolyticus suggested that the host defense mechanisms may activate 4 h after bacterial infection by means of the complement pathway and the antimicrobial peptide hepcidin [V. harveyi provided a deeper understanding of immune-related pathways acting against the bacteria [Epinephelus lanceolatus) infected with V. alginolyticus, it is believed that a TLR 5-mediated regulation leading to cytokine regulation may be involved in the immune response [Lates calcarifer) subjected to different stimuli, such as lipopolysaccharide, V. harveyi challenge, high salinity, and fasting, showed a global coordinated reaction in response to different stressors and a dramatic mucosal immune response in the intestine [RNA-Seq analysis of the half-smooth tongue sole (ilaevis)  and the ponicus)  challengvolution . A transhepcidin . Similarbacteria . In the response . The Asintestine .E. tarda live attenuated vaccine showed the upregulation of genes involved in the major histocompatibility complex (MHC)-I pathway, whereas genes associated with the MHC-II pathway were downregulated during early stages of vaccination [Dicentrarchus labrax) against V. anguillarum led to increased expression of rare transcripts, such as leukocyte immune-type receptors and cullin or supervillin [Yersinia ruckeri is a Gram-negative, rod-shaped bacterium which causes enteric redmouth disease in fishes [Acipenser schrenckii) during Y. ruckeri infection [flhD in the virulence of the bacteria [The transcriptome analysis based on RNA-Seq has been employed in few vaccination studies against bacterial infection. Zebrafish subjected to an cination . The oraervillin . Yersinin fishes ; proteinn fishes ,64. RNA-nfection  and the bacteria .Viral diseases are a paramount threat to the fish culture industry causing acute and huge economic losses. During the infection process, once a virus spreads beyond few host cells, a variety of nonspecific and specific host defensive mechanisms will be triggered, which will try to contain the virus and minimize host cell damage . DetaileAquareovirus genus of the family Reoviridae [The grass carp is an economically important candidate species for aquaculture. In recent times, grass carp farming has been severely hampered by grass carp reovirus (GCRV) infection, a dsRNA virus belonging to the oviridae . GCRV inoviridae . Howeveroviridae . Also, aoviridae .Novirhabdovirus of the Rhabdoviridae family [Oncorhynchus mykiss), a whole genome transcriptome analysis using RNA-Seq analysis was performed [Salmo salar) during ISAV infection suggested that the molecular analysis of viral segments replication has the potential to identify important immune-related genes and their responses during host–pathogen interactions [The viral hemorrhagic septicemia virus (VHSV) is a notifiable aquatic animal disease listed by OIE  infecting over 80 freshwater and marine species worldwide . VHSV bee family ,90 and rerformed . VHSV waerformed . The inferformed  and typierformed . RNA-Seqractions ,75.Oncorhynchus nerka) during PRV infection along with superinfection of IHNV suggested that the host invasion strategies of PRV and IHNV are independent of each other during coinfection. Even a robust host antiviral pathway produced during IHNV superinfection did not affect PRV multiplication [Piscine orthoreovirus (PRV) is a nonenveloped dsRNA virus belonging to the family Reoviridae and causes heart and skeletal muscle inflammation (HSMI) disease in the Atlantic salmon . This vilication . This stCyprinivirus, family Alloherpesviridae, order Herpesvirales [Cyprinus carpio koi) [Salmonid alphavirus (SAV) is an enveloped ssRNA virus from the Togaviridae family, and its infection in the Atlantic salmon may result in necrosis in the exocrine pancreas, cardiomyopathy, and skeletal myopathy . A transsvirales . KHV is pio koi) . A spleepio koi) . The sprpio koi) .Paralichthys olivaceus) revealed that heat-inactivated vaccination against VHSV modulates numerous genes involved in the acute phase response, TLR, IFN, and apoptosis pathways [Miichthys miiuy) revealed retinoic acid inducible gene-I like receptors (RLRs), like MDA5 and LGP2, that are involved in host surveillance against infection of dsRNA viruses and induce a type I IFNs response to inhibit virus replication [RNA-Seq analysis of the kidney of the olive flounder  during megalocytivirus infection in low and high temperatures showed that the temperature has a significant role in the global transcription of turbot, which in turn influences the immune system [Similarly, the orange-spotted grouper infected with the Singapore grouper iridovirus (SGIV) had a significant influence on the mitogen-activated protein kinase, chemokines, TLRs, and RIG-I signaling pathways . Megaloce system . The stuSparus aurata) (resistant species) suggested that NNV adopts a mechanism to escape the cell-mediated cytotoxicity, leading to fatal infection in the European sea bass [Gadus morhua) [Nodaviruses are RNA viruses which are categorized into three groups, i.e., alphanodavirus which infects insects, betanodavirus which infects fishes, and gammanodavirus infecting prawns . The nersea bass . Further morhua) .Similarly, previous RNA-Seq data from the grass carp during GCRV infection was used in order to check the performance of two downstream bioinformatics tools, i.e., DESeq and Cufflinks, which were used to calculate the differential expression of genes during RNA-Seq data analysis. This study provided insights into two immune-related genes, ISG15 an ubiquitin-like protein and sacsin-like gene which is likely to regulate mitochondrial dynamics in the grass carp . It is cParasites are a major concern affecting feral fish population as well as cultured fish. All of the major groups of animal parasites were reported in fish, i.e., protozoa, myxozoa, helminthic worms, and crustacean parasites. Parasites use various immune evasion strategies, like avoidance of the immune system surveillance, indirect life cycle, molecular mimicry, immune suppression, immune modulation, and manipulation of the host endocrine and neural systems to invade their hosts . StudiesCryptocaryon irritans (C. irritans) is an economically important ectoparasite which causes ich disease or cryptocaryoniasis in fish, and RNA-Seq has been used significantly to explore this disease. Immune mechanisms, such as the toll pathway, chemokine signaling, complement system, and coagulation cascades, showed profound responses during the parasite infection in the large yellow croaker [C. irritans infection. It is interesting to note that even a low, nonlethal infection of C. irritans could induce a significant immune response in fish; in contrast, an immunosuppression-like condition was noticed in the large yellow croaker with a fatal infection [C. irritans [C. irritans with the objective of identifying potential vaccine candidates. The analysis of the tomont stage of C. irritans under low temperature helped in the identification of several genes required for the parasitic stage to enter a deeper dormancy state and survive low temperatures. These genes might be considered as potential candidate genes to develop diagnostic and control measures for cryptocaryoniasis [C. irritans which could be considered as major targets for vaccines and drug development, respectively [Caligus rogercresseyi as well as from the adult male and female will serve as a valuable tool for future genomic studies to understand host–parasite interactions in the salmon louse [ croaker . In addinfection . There wirritans . Two stuyoniasis . Similarectively . Similaron louse .Gasterosteus aculeatus) was infected with three different genotypes of the trematode parasite Diplostomum pseudospathaceum, the genetic variation in parasitic worms influenced the mechanisms by which the host immune system reacted to an immunological threat [When the three-spined stickleback  showed an exacerbated local immune response in the pyloric caeca, whereas an inadequate activation of the adaptive immunity was observed in the spleen and kidney as a result of the failure in bridging between the innate and adaptive immune responses [E. scophthalmi showed the vital role of the IFN-mediated immune response during the early phase of the disease [Tetracapsuloides bryosalmonae  is the causative agent of proliferative kidney disease which significantly affects both farmed and wild salmonid fishes in Europe and North America, causing significant economic losses, and endangers the wild fish population [T. bryosalmonae and its invertebrate host Fredericella sultana, and only a few expressed sequence tags have been deposited in the National Center for Biotechnology Information (NCBI) GeneBank database. Recently, it has been demonstrated that T. bryosalmonae can complete the development and produce mature spores only in the brown trout but not in the rainbow trout [T. bryosalmonae.RNA-Seq analysis of the large yellow croaker infected with the intestinal myxozoan parasite esponses . Similar disease . The myxpulation . There iow trout ,119,120.Aphanomyces invadans (A. invadans) and Saprolegnia parasitica (S. parasitica) are endemic to freshwater habitats and belong to the water molds or oomycetes group. These two oomycetes are responsible for significant economic losses in the aquaculture industry [A. invadans is known to cause the epizootic ulcerative syndrome across fish species and could possibly invade the host by means of some extracellular proteases [S. parasitica causes the saprolegniasis disease, a major threat to the salmonid farming industry. Only one study has exploited RNA-Seq-based transcriptome analysis to understand S. parasitica. RNA-Seq analysis of four life stages and a cell culture-based time-course study revealed that the kinome of S. parasitica has a significant function during infection. Furthermore, several putative proteins expressed in preinfection life stages may help the oomycete to interact with the host [industry . A. invaroteases . Similarthe host .Gymnocypris przewalskii) [Gymnocypris przewalskii ganzihonensis) [Culter alburnus) [Takifugu rubripes) [Callorhinchus milii) [Plecoglossus altivelis) were employed to design a customized microarray, which was used to test the effects of recombinant LECT2 on the macrophages of ayu fish [Fish is an evolutionarily primitive animal among vertebrates, and the immune system of fish is highly complex. The immune system works continuously to protect the host from invading pathogens. Transcriptome analysis based on RNA-Seq is now being used to understand the immune system and its evolutionary significance in normal fish without infections . The immwalskii) , the schonensis) , the toplburnus) , and thelburnus)  were stuubripes) . Similarubripes) . Also, Rubripes)  and a res milii) . Transcrayu fish . Recentlayu fish . Also, tayu fish .Misgurnus anguillicaudatus), which revealed a complex immune mechanism involving chemokine signaling, leukocyte transendothelial migration, and T cell receptor signaling [Mucosal organs, such as the gut, skin, and gill, are constantly exposed to the external environment and could be considered as the main route for pathogen entry in fish . Hence, ignaling . Similarignaling . Nutritiignaling .Melanotaenia duboulayi) under high-temperature stress [Anguilla japonica) when compared to lower salinity [Spatholobus suberectus, Phellodendron amurense, and Eclipta prostrate, after dietary supplementation to the tiger grouper. The experiment interestingly revealed that S. suberectus was immunostimulatory, whereas P. amurense and E. prostrate were immunosuppressive [Abiotic factors, such as temperature, crowding, salinity change, sunlight, and oxygen levels, are found to exhibit a significant influence on the immune system of fish. RNA-Seq-based transcriptome profiling unveiled alterations in immune genes of the crimson-spotted rainbowfish (e stress , of the e stress , of the e stress , and of e stress . Under he stress . Similarsalinity . Lipopolsalinity . Plant-bsalinity . RNA-Seqpressive . This waIn this review, we have summarized the applications of RNA-Seq technology for a deeper understanding of pathogenic diseases and defense mechanisms in fish. Being qualitative as well as quantitative, this technology has led to the identification of many molecular actors and their patterns of expression in a limited time-frame, laying the foundation for further studies in the mechanisms of host–pathogen interaction. This technology has also aided in testing the efficacy of vaccines ,44,74, tApart from this, RNA-Seq-based transcriptome analysis of fish has its own limitations compared to its mammalian counterpart. During evolution, genome duplication has determined a large variation in the genes of fish compared to other animal groups , and theThe use of RNA-Seq to analyze transcriptome changes started at the end of the last decade and, day by day, its application in the field of fish health is in the log phase. This technique is highly dynamic and evolves based on new challenges and research objectives. In the past few years, ScRNA-Seq protocols have gained momentum, rendering transcriptome information from a single cell and thus avoiding the limitations caused by heterogeneous cell populations . However