The current Covid-19 pandemic has pointed out some major deficiencies of the even most advanced societies to fight against viral RNA infections. Once more, it has been demonstrated that there is a lack of efficient drugs to control RNA viruses. Aptamers are efficient ligands of a great variety of molecules including proteins and nucleic acids. Their specificity and mechanism of action make them very promising molecules for interfering with the function encoded in viral RNA genomes. RNA viruses store essential information in conserved structural genomic RNA elements that promote important steps for the consecution of the infective cycle. This work describes two well documented examples of RNA aptamers with antiviral activity against highly conserved structural domains of the HIV-1 and HCV RNA genome, respectively, performed in our laboratory. They are two good examples that illustrate the potential of the aptamers to fill the therapeutic gaps in the fight against RNA viruses. The current global pandemic caused by SARS-Cov-2 has revealed the fragility of our global health security system. Researchers have been warning governments about the risk of different heath threats we face today, and this outbreak has clearly shown that no country, not even the so called first world, is safe and protected against a sanitary emergency. Among other deficiencies, one of the major limitations to fight against the current viral infection has been the lack of specific therapeutic agents, which is a common fact for many other infections caused by RNA viruses . Researchers have traditionally devoted many efforts to searching for the magic molecule or therapeutic strategy that would allow fighting efficiently against these tiny organisms that so easily put the most evolved living being in check.RNA viruses store all the required information for the completion of the infectious cycle in their RNA genome. In addition to the information that encodes the viral proteins, the genome includes the information required to efficiently sequester and utilize the cellular machinery and all the information involved in the regulation of the viral processes. RNA viruses have developed different molecular strategies to bear all the information, compacting it in different hierarchical levels of coding within the RNA genome ,2,3. ThuIt was in 1990 that two independent publications from the groups of Jack Szostack and Larry Gold set the basis for the development of the currently known aptamer technology ,5. The gThe potential of this technology was envisioned quite early, and the therapeutic application of aptamers for a wide variety of diseases was proposed. It was in 2004 that the FDA approved the first aptamer to be used as a specific drug. This aptamer, which was named Pegaptanib and commercialized as MACUGEN, was meant for the treatment of the wet age-related macular degeneration ,16,17. N12 to 1016 variants, is subjected to the SELEX procedure, which consists of iterative cycles of exposition to the target molecule to allow their binding to the target, separation of those molecules capable of binding and their further amplification. This procedure allows the progressive enrichment of the population in winner molecules for specific binding to the desired target. Those winner molecules are named aptamers. Independently of the application of the aptamers and the nature of their target, all reported aptamers have been identified following a common experimental strategy known as SELEX ,18, whicThe researcher can control at any time the conditions of the selection and recovery steps during the process. Usually, the main goal is to identify the selection to yield aptamers that exhibit high affinity by a desired target, but the researcher can also pursue the selection of aptamers that achieve stable binding by the imposed conditions. It has been shown that aptamers may bind the ligand molecule with efficiencies that even may exceed that of antibodies for their antigens .Aptamers show some important advantages , includiSpecificity of aptamers is determined by both their sequence and their three-dimensional folding. Further, aptamers recognize specific functional groups of the target molecule in a precise spatial distribution. Therefore, aptamers are good candidates to target complexly folded targets. Consequently, in contrast to other nucleic acid based strategies , aptamers are postulated as excellent candidates to directly interfere with the functioning of essential structural domains of viral RNA genomes.Targeting viral proteins is the common strategy that has been followed in most attempts aimed to develop efficient antiviral agents regardless of the drug to be used. Aptamers selected against a variety of viral proteins have also been reported in the literature. Those studies account for the achievement of a range of antiviral activity levels . Unfortunately, up to now such a strategy has been found to be very inefficient, yielding in most cases unacceptable therapeutic results. Even in the best scenarios, the achievements that have occurred over the years of enormous economic and human resources investments are unfortunately not enough to compensate for the unacceptable number of human lives and the consequences on the health and well-being of humans that viral infections take, among other consequences. This is evident in its maximum expression in a pandemic situation like the one we are currently experiencing. Therefore, it is widely accepted by the scientific community the need of exploring alternative viral targets and developing of new therapeutic antiviral strategies . TargetiIn both cases we followed a common SELEX strategy . BrieflyThe pool of RNA oligonucleotides was binding-challenged against the immobilized target molecule. Then, only oligonucleotides capable of binding to the target RNA were retained. After their selective elution and amplification by RT-PCR, the new generated pool was subsequently introduced in a new round of selection. This was repeated until rendering the most efficient binders to the viral genomic fragment . We applied the above experimental strategy to isolate aptamers against the 5′UTR of the human immune deficiency type 1 virus (HIV-1) . For thi15 oligonucleotide variants, resulting from the randomization of 25 contiguous nucleotides. The selection cycle was repeated up to 14 times ..25].The innovative combination of a SELEX procedure with a bioinformatics analysis allowed the identification of a common 16 nt-long structural element, consisting of an octanucleotide of fixed sequence exposed in a loop motif closed by 4 bp stem of variable sequence that retained the full antiviral activity. It served as base for the design of the, to our knowledge, smallest aptamer ever described. It is worth noting that the minimum molecular size of the aptamers is an important limitation. The size is imposed by the need of carrying flanking fixed sequences required for the amplification steps of the SELEX procedure. The application of the bioinformatics analysis provides unique means of identifying the minimum domain responsible of the entire aptamer activity.The hepatitis C virus (HCV) has been another favorite virus for the development of nucleic acids-based antiviral strategies. The organization and structure of its RNA genome have been extensively studied . It cont18 RNA variants, resulting from the randomization of a stretch of 30 nts, was introduced in the selection process. The resulting variant molecules after six selection rounds were classified in up to five families, defined by common selected sequence motifs. As expected, each common sequence possessed its complementary counterpart within the viral CRE [We applied the previously described SELEX strategy to identify RNA aptamers against a 194 nt-long subgenomic RNA comprising the CRE . A populiral CRE . The preiral CRE . InteresIn an independent series of experiments, we isolated a new collection of RNA aptamers conjugated with ribozymes that specifically targeted the translation-essential HCV IRES , which is located at the 5′ end of the RNA genome . Those RAll together the above reported results point the great antiviral potential of targeting essential structural RNA domains of the HCV genome.Aptamers offer a potential means for the development of efficient antiviral drugs. Their mechanism resides in the recognition of functional groups in a specific structural context in the target molecule, similar to antigens recognition by antibodies. Viral RNA genomes store essential information for the completion of the viral propagation cycle in structural RNA elements. These elements are grouped in structurally complex domains and regions that coincide with the highest conserved regions of the highly variable viral genome among different viral isolates. The preservation of their structure is essential for the proper functioning of each of these elements, offering an excellent scenario for fighting infections caused by RNA viruses. Aptamers have proven to be capable of interfering with the activity of these essential domains by competing with the interactions they are involved in or by modifying their structure. Different groups have already reported aptamers that induce potent inhibition rates of a variety of RNA viruses by targeting conserved genomic RNA domains. Therefore, aptamers emerge as real molecular tools to fight viral infections.


Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in  Among tn cycles . Differen cycles ,3,4. Addn cycles .Despite their well-recognized roles in viral multiplication cycles, some of the RNA secondary structures are difficult to study because they often reside in coding sequences for important viral proteins, including RdRPs and auxiliary replication proteins (ARPs). For instance, many a (+) RNA virus encode RdRP by extending ARP at the C-terminus by avoiding the stop codon of the latter through either translational read-through or frame-shifting. Such read-through and frame-shifting events are tightly regulated through highly conserved RNA structures that simultaneously encode part of the RdRP ,7. For RNicotiana benthamiana as hosts [Here we report an approach to uncouple the protein-coding capacities of viral gRNA from the RNA secondary structures it folds into, using turnip crinkle virus (TCV) as our model. TCV is a small (+) RNA plant virus that counts model plants Arabidopsis and as hosts ,9,10,11.as hosts ,9,12.Both p28 and p88 are translated directly from TCV gRNA, with the latter resulting from programmed translational read-through of the p28 stop codon. This translational read-through event is facilitated by a well-characterized, highly conserved RNA secondary structure known as a recoding stimulatory element (RSE), in coordination with another 3′ terminal structure through a long-distance kissing loop interaction ,7. RSE atrans, thus obviating the need to maintain the protein-coding capacity and the structures that regulate protein translation within the p28/p88 coding region of TCV genome. This alternative approach allowed us to confirm a replicational role of a previously characterized structure, known as internal replication element (IRE) [In the current study, we assessed the translation-independent role of several previously identified structure/sequence elements within p28/p88 coding sequence by providing the p28/p88 proteins in nt (IRE) . More imThe constructs TCV_sg2R, Core35S::p88-2HA, and 2 × 35S::p28 (tag-free) were described in previous studies ,12,14. AAgrobacterium tumefaciens strain C58C1 with electroporation [Agrobacterium suspensions were mixed and delivered into N. benthamiana leaves, as described in [Agrobacterium strain was included in all combinations to alleviate RNA silencing-mediated mRNA degradation.Upon verification, the constructs were introduced into poration . In mostribed in ,12,14. AN. benthamiana leaves, using the Direct-zol RNA Miniprep kit . To ensure consistency, four equivalent leaf sections derived from infiltrated leaves of four different plants were pooled before RNA extraction. The RNA was then quantified with NanoDrop and subjected to Northern blotting, as described in [Total RNA was extracted from agro-infiltrated ribed in ,14. Quantrans, using a transient expression construct, Core35S::p88-2HA , leaving the coding sequence of p28 intact  encompassed two previously reported structures—Fa (nt 816–847) and RSE (nt 816–905), with the former being the front half of the latter ,15 FiguA. This 8trans, the gRNA levels of the 813UAA mutant were 40–70% of TCV_sg2R in different experiments  The first 11 positions of the RSE contain seven invariable nts highly conserved among more than 35 viruses of To determine whether the seven conserved nts contribute to TCV gRNA levels in infected cells, we created m2-1, in which all of the conserved nts were mutated into their complementary residues D,E. TherMindful of the previous study showing that the CCCC motif at the bottom-right of RSE engaged in a stable pseudoknot with GGGG near the top of RSE (nt 860–863)—and this pseudoknot was needed for efficient translational read-through —we next The results with mutants m2-2 and m2-3 suggested that the specific nt sequence altered in m2-2 A was cruThe reductions in TCV gRNA levels by the mA2 mutations, as well as the pseudoknot mutations, while substantial, were short of the near complete loss of the ΔFa* mutant. We hence moved to test another known sequence motif inside RSE, namely the asymmetric loop roughly in the middle of RSE , by changing the codon position #37 of p28 from AUG to UAG, in the 813UAA background , Δ1208–2197 (deleting 990 nt), and Δsg1Pro (deleting 195 nt). Although the gRNA accumulation levels of these mutants were modest in comparison to the TCV_sg2R + p88 control, they were similar to another control replicon (813UAA + p88) incapable of producing any p88 by itself, suggesting a slight advantage of cription . Thus, tThird, structures implicated in translational read-through can have additional roles in genome replication. This is best illustrated by the pseudoknot structure residing in the RSE of TCV, which was found earlier to be needed for efficient translational read-through. We found that it was also needed for efficient accumulation of TCV gRNA, probably through its participation in genome replication. It is possible that this dual function could be related to coordinating translational read-through with genomic minus-strand synthesis, which are directionally opposing processes on the genomic RNA. In this scheme, read-through would unfold the pseudoknot needed for full-length genome minus-strand synthesis, thereby inhibiting this competing process. The maintenance of sgRNA accumulation when the pseudoknot is disrupted suggests that the inhibition may be specific for synthesis of full-length genomic minus-strands. RNA-mediated coordination of these two processes occurs in tombusviruses, where formation of the long-range interaction between the RSE and 3′ UTR required for RdRP read-through concomitantly prevents formation of an alternative RNA structure in the 3′UTR needed for minus-strand RNA synthesis . More detrans) than in the 813UAA backbone (needing only p88 in trans). Finally, the RSE element could be under dynamic regulation through the folding of the alternative Basal structure that encompasses the Ff element within the p28 coding region, as mutating or deleting Ff appeared to also affect the robustness of TCV replication. Again, the Basal structure appears to be less critical when the replicon genome produces its own p28, as the m2-1 mutant would be expected to have disrupted the FfFa stem extensively, yet could replicate to 70% of its 813UAA parent (The integrity of the middle portion of the RSE (stem I) may also serve as an additional sentinel for quality control of TCV genome. It is possible that involvement of stem I in TCV replication could entail collaboration with p28 translated from the same RNA, because the reduction in gRNA levels caused by mA2 mutations was much less pronounced in the p28TS replicon backbone (in need of both p28 and p88 in A parent .trans-provided p28 and p88 was inefficient compared to that of TCV replicons encoding their own p28 and p88. Multiple factors could have contributed to this inefficiency. First, both p28 and p88 were shown previously to repress TCV replication when overexpressed [cis-production of these replication proteins may indeed be favored by the replication process [Finally, we wish to address why the replication of TCV mutants with xpressed ,12. Assu process ,22. Neve


Cardiovascular diseases (CVDs) are of multifactorial origin and can be attributed to several genetic and environmental components. CVDs are the leading cause of mortality worldwide and they primarily damage the heart and the vascular system. Non-coding RNA (ncRNA) refers to functional RNA molecules, which have been transcribed into DNA but do not further get translated into proteins. Recent transcriptomic studies have identified the presence of thousands of ncRNA molecules across species. In humans, less than 2% of the total genome represents the protein-coding genes. While the role of many ncRNAs is yet to be ascertained, some long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been associated with disease progression, serving as useful diagnostic and prognostic biomarkers. A plethora of data repositories specialized in ncRNAs have been developed over the years using publicly available high-throughput data from next-generation sequencing and other approaches, that cover various facets of ncRNA research like basic and functional annotation, expressional profile, structural and molecular changes, and interaction with other biomolecules. Here, we provide a compendium of the current ncRNA databases relevant to cardiovascular research. Cardiovascular diseases (CVD) are ailments of the heart and the circulatory system and include a variety of highly prevalent disorders such as coronary artery disease (CAD), atherosclerosis (AS), atrial fibrillation (AF), ischemic heart failure (IHF), and congenital heart disease (CHD). Although CVDs are multifactorial in their origin, the most common causes are genetic and environmental changes. CVDs are the prime trigger of premature mortality worldwide, accounting for over 17.9 million deaths, as of 2016 [Non-coding RNAs (ncRNAs) are a type of RNA molecule, which are not translated into proteins. In humans, less than 2% of the genome correlates to the coding region, despite a transcription rate as high as even 93% ,5. More Increasing studies have indicated ncRNAs to have a role in the progression of cancers, cardiovascular diseases, and other complex disorders, serving as a regulator of numerous cellular processes . AdditioA key characteristic in support of ncRNAs is that they are easily detectable biomarkers in body fluids , etc.) promoting the use of minimally-invasive techniques such as liquid biopsies ,16. A reThe following ncRNAs were used to test the properties of the respective noncoding RNA databases. All of these ncRNAs are relevant to cardiovascular diseases and are detailed in the following section. Myosin heavy chain associated RNA transcript (MHRT) is a gene encoding a spliced form of a non-coding RNA, acting as a cardioprotective agent in the heart. This lncRNA is conserved in nature, seen also in mice (Mhrt), where they have been shown to serve as a potential biomarker for cardiac hypertrophy and heart failure ,15,25,26NONCODE was originally published in 2005, serving as the first unified resource on ncRNAs . Since tRNAcentral is an exhaustive ncRNA database containing a collection of different types of ncRNA sequences from a wide range of species . LauncheNPInter is a repository of experimentally-verified functional interactions between the ncRNAs and other biomolecules like genomic DNA, RNA, and protein. NPInter was first published in 2006 and subsequently updated periodically ,52,53. TmiRbase is a repository of miRNA sequences and annotations, originally published in 2002 as the microRNA registry ,58 and sexoRBase is a repository of lncRNAs, circRNAs and mRNAs from RNA-seq analysis of human exosomes . The datpiRBase is an exclusive database for all information about PIWI-interacting RNAs, a small subset which is highly expressed in germline tissues and is being increasingly shown to have a role in epigenetic and post-transcriptional modulations. The database was originally launched in 2014 and has subsequently been updated in 2018, covering 21 species and 173 million piRNA ,66. The CVDncR is a database of manually collected data of non-coding RNA, especially microRNA, long non-coding RNA and circular RNA, related to cardiovascular diseases . The datThis unique database provides a landscape of the transcription in human heart tissue . RNA-seqApart from the resources above, there are other unique databases such as CRISPRlnc, RISE, RNAlocate, and MNDR which serve specific requirements. CRISPRlnc is a manually curated database of validated CRISPR/Cas9 sgRNAs for lncRNAs from eight species. The database comprised of 2100+ sgRNAs for 300+ lncRNA sequences . RISE isENCORI (The Encyclopedia of RNA Interactomes), previously known as starBase, is also a useful resource which has now been updated to include over 7 million interactions between RNA subtypes, mined from publicly available big-data experiments ,77. The Despite the existence of numerous resources in support of noncoding RNA research in cardiovascular genetics, there remain questions that are yet to be decoded. Some of the areas, which will require future attention include the evolutionary conservation of lncRNAs in heart tissue and lncRNA-circRNA associations among others. The recent advances in the field of high-throughput sequencing have led to sophisticated methods for RNA detection, such as total RNA sequencing and single-cell sequencing. This, in turn, has drastically increased the discovery rate of ncRNAs, particularly lncRNAs and circRNAs in recent years. Keeping in mind recent findings that these ncRNAs have an extended role in disease initiation, progression, and general pathophysiology, RNA molecules have now emerged as a strong therapeutic candidate against CVDs and such databases will prove a pivotal resource for researchers working in this domain.


Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals. Potyviridae, the most important group of plant RNA viruses. Using plum pox virus  as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses.The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family  Such7–cination . Remarkacination , 12, andpromised , 14. Givpromised , 14.Despite the importance of plant viruses, and the enormous negative consequences of viral diseases for sustainable agriculture and food security, much less is known about their genome restrictions at the level of dinucleotide composition. In fact, the low frequency of CpG and UpA in the genomes of plant-infecting RNA viruses was noticed a long time ago , 6 and rin planta than its parental control. Finally, aiming to understand the reasons underlying the UpA-based viral attenuation, we found that this restriction (i) is independent of the classic antiviral RNA silencing pathway, (ii) occurs at the level of RNA molecules, and (iii) also influences the accumulation of RNA polymerase II-transcribed RNAs.In this study, we manipulated the genome of plum pox virus (PPV) to find that an increased frequency of either CpG or, especially, UpA has a negative impact on viral fitness. A further comprehensive manipulation of UpA frequencies showed a dose-dependent effect, which extended to a mutant in which the UpA frequency was even lower than in the wild-type (WT) virus; such a mutant displayed greater fitness Potyviridae, the most abundant and socioeconomically relevant group of plant RNA viruses. We followed the Karlin and Mrazek criteria to estimate over- and underrepresentation of the 16 dinucleotides   and less variable than that of CpG (0.632 ± 0.066 for UpA versus 0.707 ± 0.146 for CpG)  , howeverfor CpG) . The UpAfor CpG) .10.1128/mBio.02818-19.5TABLE S1Potyviridae. Download Table S1, PDF file, 0.1 MB.Odds ratio for members of the family Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Alphaflexiviridae , size of the RNA molecules , and virus particle structure , we envisage that the observed dinucleotide bias is a general feature of plant positive-strand RNA viruses.UpA and CpG dinucleotide restrictions are not an exclusive feature of potyvirid genomes, since equivalent analyses of other families of plant single-stranded (ss), positive-sense RNA viruses, such as iviridae , Closteroviridae , and Brooviridae , show thleotides  and S4. 10.1128/mBio.02818-19.6TABLE S2Alphaflexiviridae. Download Table S2, PDF file, 0.1 MB.Odds ratio for members of the family Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02818-19.7TABLE S3Closteroviridae. Download Table S3, PDF file, 0.1 MB.Odds ratio for members of the family Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02818-19.8TABLE S4Bromoviridae. Download Table S4, PDF file, 0.1 MB.Odds ratio for members of the family Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Potyviridae family, Potyvirus genus) (We selected PPV (s genus)  as a mods genus) . First, s genus) . Based o10.1128/mBio.02818-19.1FIG S1y-axis scale) has been superimposed to identify areas of thermodynamically favored base pairing. (B) Images taken with an epifluorescence microscope of upper noninoculated leaves, at 10 and 20 dpi, from plants infected with the indicated permuted viruses (_perm) and their parental control (SD-PPV). Bars, 5 mm. (C and D) PPV CP-specific immunoblot analyses of extracts from upper noninoculated N. benthamiana leaves  infected with the indicated viruses at 10 (C) and 20 (D) dpi. Membranes stained with Ponceau red showing the RubisCO large subunit were included as loading controls. Bar graphs showing the mean ± SD (n = 4) of immunoblot signals in arbitrary units are shown on the right . Different letters indicate significant differences (P < 0.05), by one-way ANOVA and Tukey’s HSD test. Download FIG S1, JPG file, 0.4 MB.Scanning the PPV genome to identify target regions suitable for manipulation of dinucleotide frequencies. (A) Scan of synonymous variability  among 61 aligned nonredundant (>1% divergence) PPV sequences to identify regions of suppressed variability indicative of overlapping reading frames and structured RNA elements. A plot of MFED  . It is worth mentioning here that these modifications, as well as those introduced to change dinucleotide frequencies (see below), did not significantly alter codon usage (see the codon adaptation index [CAI] column in on (dpi) . Consiston (dpi) C and D. 10.1128/mBio.02818-19.9TABLE S5Table S5, PDF file, 0.1 MB.Composition of different PPV-derived fragments. Download Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the First, we evaluated the effect of CpG and UpA dinucleotide compositional modification of segment F4 in mutants, using agroinfiltration as the inoculation method  see Mat. At 10 aPrunus trees. We agroinoculated Prunus persica, a natural host of PPV, with SD-PPV and derivative viruses having altered dinucleotide frequencies in F4. Analogously to N. benthamiana plants, the F4_UpA_high mutant was unable to reach upper noninoculated leaves, whereas SD-PPV and the remaining mutants accumulated at comparable levels in these tissues, as observed by immunoblotting at 15 dpi , suggesting that attenuation induced by dinucleotide frequency changes was independent of the inoculation method. We also showed that some aspects of the attenuation may be host dependent. The SD-PPV chimera can infect t 15 dpi . However persica ; hence, N. benthamiana was observed in mutants with equivalently mutagenized sequences in the F3 region, a different 1-kb fragment of SD-PPV (N. benthamiana plants (N. benthamiana was as follows: SD-PPV ≃ CpG_low ≃ UpA_low > CpG_high ≫ UpA_high.A similar pattern of attenuation of F4 mutants in f SD-PPV . An incra plants E and F. a plants A and B. a plants  to D. Fo10.1128/mBio.02818-19.2FIG S2N. benthamiana leaves infected with the indicated mutant viruses (F3_) and their parental control (SD-PPV). Inoculated leaves at 6 days postinoculation (dpi) and upper noninoculated leaves at 10 and 20 dpi are shown. Bars, 5 mm.  PPV CP-specific immunoblot analyses of extracts from upper noninoculated N. benthamiana leaves  infected with the indicated mutant viruses (F3_) and their parental control (SD-PPV) at 10 and 20 dpi. Membranes stained with Ponceau red showing the RubisCO large subunit were included as loading controls. Bar graphs showing the mean ± SD (n = 4) of immunoblot signals in arbitrary units are shown on the right . Different letters indicate significant differences (P < 0.05), by one-way ANOVA and Tukey’s HSD test. For this experiment, all viruses were inoculated by agroinfiltration, with the exception of F3_UpA_high, whose infectious cDNA plasmid was unable to transform Agrobacterium tumefaciens due to unknown reasons. Hence, the inoculation of this particular mutant and its parental control was carried out by biolistics in an independent experiment (E and F). Download FIG S2, JPG file, 0.4 MB.Functional analysis of CpG and UpA dinucleotide constraints in another segment of the SD-PPV genome.  Images taken with an epifluorescence microscope of Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the From the experiments presented so far, we conclude that the increase of either CpG or UpA frequencies in two different segments of the SD-PPV genome results in modest and strong virus attenuation, respectively. The difference in degree of attenuation observed between UpA_high and CpG_high mutant viruses correlates positively with the greater restriction for UpAs observed in potyvirid genome sequences (see above). For this reason, we decided to concentrate further efforts on understanding the relevance of this particular dinucleotide for viral fitness.N. benthamiana plants with these mutants and SD-PPV as the control, and infections were followed under UV light. Although all mutants displayed similar levels of fluorescence in inoculated leaves at 6 dpi , and this may have contributed to the noticeably lower degree of attenuation. To investigate the dose dependence of UpA addition on viral fitness, we divided the F4_UpA_high sequence into two halves, F4.1 and F4.2 , containthamiana  and C, athamiana  and D, tN. benthamiana plants inoculated by biolistics, since this method allowed precise calculation of the proportion of each infectious cDNA clone in the inoculum. Two independent mixes (mix 1 and mix 2) as biological replicates were prepared with twice the amount of parental pLX-SD-PPV, to provide a more stringent test of fitness enhancement. Analysis of chromatogram peaks of sequenced PCR products from inocula confirmed that the amount of pLX-SD-PPV in these mixes was higher than that of pLX-F3-4_UpA_low  than the F4.1_UpA_high variant    were agr4 plants . In cont4 plants . The est4 plants  agreed wN. benthamiana leaves by agroinfiltration. In this case, transcription is under the control of the strong 35S promoter, which drives the production of RNAs by RNA polymerase II in the nucleus of infiltrated plant cells. To allow their detection by immunoblotting, the F4-derived proteins were tagged with a 4×MYC epitope at their N termini. These proteins were coexpressed with pothos latent virus (PoLV) P14, a potent RNA silencing suppressor, aiming to avoid the well-known effect of the classical RNA silencing over all transgenes. The estimation of protein accumulation by immunoblotting against the 4×MYC tag at 7 dpi showed a much lower expression of 4×MYC-F4 when the protein was expressed from the UpA-rich RNA   were trarich RNA . We thenrich RNA . As thesrich RNA , this rerich RNA , the lowrich RNA , the 4×Mrich RNA .10.1128/mBio.02818-19.4FIG S4N. benthamiana leaves by agroinfiltration. Tested samples correspond to those analyzed in n = 2 plants). For comparison, we considered the average for F4_WT to be 1. Download FIG S4, JPG file, 0.05 MB.qPCR to estimate the amount of DNA introduced in Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CpG/TpA constraints were reported a long time ago in animal and plant genomes –26, but –N. benthamiana, P. persica, and A. thaliana), suggesting that it could be a general feature of any plant-virus combination. Among the results with UpA-modified PPV variants, those of the competition experiment are the most intriguing. Replication enhancement was indeed previously observed by reduction of UpA frequency in echovirus 7 (Picornaviridae family) in human cells  in three tested hosts  attenuation of F3_CpG_high and F4_CpG_high PPV variants shown in the current study suggest that cellular pathways underlying dinucleotide restriction in plants may differ from those operating in animals.On the other hand, the attenuation of CpG-rich PPV was much less pronounced in in vitro with a macrophage cytoplasmic RNase as long ago as 1989 by Beutler and collaborators, as they noticed that the presence of TpA is particularly constrained in DNA destined to produce RNA that will accumulate in the cytosol   overlap only spatially in the cytoplasm, we also speculate that the proposed restriction operates in this compartment. In fact, the idea that a specific RNase hydrolyzes UpA-rich RNAs in the cytoplasm has been postulated and studied , tRNAs) . Hence, Irrespective of mechanisms, however, the demonstrated impact of dinucleotide frequency modification in the genomes of plant viruses (and the great stability of this effect over the time) opens the possibility of developing systematic approaches based on dinucleotide manipulation to control viral fitness for different purposes. Cross-protection , virus-induced gene silencing, and potential enhancement of heterologous expression through compositionally modified virus mutants, vectors, and transgenes are all areas that may be exploited in the future.xy) was calculated with the formula ORxy = xyf/(xf × yf) (where f is frequency) by using the program Composition Scan in the SSE package v1.3 . Mean MFED (minimum folding energy differences) values for each fragment were plotted against the midpoint of each fragment to localize areas of sequence-order-dependent RNA secondary structure. Synonymous sequence variability was determined by measurement of mean pairwise distances between successive fragments of 300 bases, with 30-base increments between fragments, using the program Sequence Scan in the SSE package.A total of 156 coding region complete sequences of PPV were downloaded from GenBank. Sequences were filtered to 1% nucleotide divergence to leave 61 sequences available under the following accession numbers: N. benthamiana codon usage, which was evaluated by using the tool available at the website http://genomes.urv.es/CAIcal/  of around 1 kb each . For per/CAIcal/ . All of N. benthamiana, a model plant, and P. persica, a natural host of PPV (N. benthamiana and (ii) PPV-D in Prunus persica, so that little differences in virus accumulation due to the introduced modifications should be observed and quantified more easily in both hosts. The infectious cDNA clone pLX-SD-PPV was built by replacing the ScaI/SalI 4,767-bp fragment from pLX-R-PPV with the equivalent fragment from pICPPV-5′BD  and then excised from the provided intermediate plasmids with the same restriction enzymes that were later used for exchanging the equivalent wild-type segments in pLX-SD-PPV  and pLX-The sequences of all primers used for this study are listed in 10.1128/mBio.02818-19.10TABLE S6Table S6, PDF file, 0.05 MB.List of primers used in this study. Download Copyright © 2020 González de Prádena et al.2020González de Prádena et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Wild-type, UpA_low, and UpA_high versions of the F4 segment were amplified by PCR with the primer pair 3513/3514, which adds a stop codon. These products were cloned with Gateway technology in the destination vector pGWB718  , using pN. benthamiana and P. persica GF305 and 8-h-light/16-h-dark cycles at 21°C for A. thaliana. Leaves of 4-week-old plants were infiltrated using Agrobacterium tumefaciens C58C1 strains carrying the indicated plasmids, as previously described (600) of 0.05 for infiltrations in N. benthamiana, an OD600 of 0.5 for infiltrations in P. persica, and an OD600 of 1.0 for infiltrations in A. thaliana. When required, N. benthamiana plants were inoculated by bombardment with microgold particles coated with DNA of the indicated plasmids by using a Helios gene gun system (Bio-Rad), as previously described . Virus-derived GFP fluorescence was observed in a Leica MZ FLIII stereomicroscope (Leica Microsystems), and images were acquired as described previously , by following the manufacturer’s instructions. Second, around 500 ng of total RNA was subjected to reverse transcription with the Moloney murine leukemia virus (M-MuLV) enzyme (New England BioLabs) and random hexanucleotides as primers, and the so-generated cDNAs were then used as the template to amplify the whole F4 segment (primer pair 3463/3345). Finally, PCR products were Sanger sequenced (Macrogen Europe).N. benthamiana plants infected with the indicated viruses and harvested at 21 dpi. The first amplicon spans a region of segment F4.1 , whereas the second one spans a region of segment F4.2 . These PCR products were then subjected to two additional rounds of PCR, with a low number of cycles, to attach the appropriate adaptors and barcodes at their 5′ and 3′ ends. Paired-end sequencing (2 × 300) was done with a MiSeq reagent kit v3 on a MiSeq sequencing platform (Illumina) by following the manufacturer’s instructions.Two amplicons were generated by RT-PCR, as explained above, from upper noninoculated leaves of https://sourceforge.net/projects/bbmap/). These alignments were analyzed with SAMtools (H = −∑i (pi × ln pi); where H is the Shannon entropy at a given position, and pi is the probability of having any of the four nucleotide (i) at that position], and used the average of H to estimate variability per amplicon . After centrifugation at 13,000 × g for 10 min, samples were boiled for 5 min at 95°C. Proteins were separated in SDS-PAGE gels (12% acrylamide) and electroblotted onto a nitrocellulose membrane. The CP from PPV was detected with a specific rabbit serum used as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) as the secondary reagent. MYC-tagged proteins were detected with a specific anti-MYC monoclonal antibody (AbMART) and HRP-conjugated sheep anti-mouse IgG (Sigma) as the secondary antibody. Immunostained proteins were visualized by enhanced chemiluminescence detection with Clarity ECL Western blotting substrate (Bio-Rad) in a ChemiDoc system (Bio-Rad). Band intensity in arbitrary units was estimated by using Image Lab software (v.6.0.0), with the signal of one selected sample considered as the reference. Ponceau red staining of membranes was used to check the global protein content of samples.Crude extracts were obtained from Total RNA was isolated from the indicated ground leaf tissue, and cDNA was synthesized as described above. The abundance of specific transcripts was measured by probing the cDNA by quantitative PCR using EvaGreen master mix (Solis Biodyne), in a 7500 real-time PCR system (Applied Biosystems), with primers 3519/3520 (MYC) or 3527/3528 (NOS). The transcript levels between samples were normalized against the expression of PP2A housekeeping gene  using prMiSeq raw data are available in ArrayExpress under accession number E-MTAB-8399.


Seven strains of coronavirus family can infect humans and can cause different signs ranging from cold with major symptoms such as fever and sore throat to upper and lower respiratory tract infections resulting in pneumonia, severe respiratory tract infection and even death. These seven strains include HCoV-229E, HCoV-OC43, SARS-Co- V, human coronavirus NL63, human coronavirus HKU1, MERS-CoV and SARS-CoV-2, known as 2019-nCoV or “novel corona virus 2019” 3.Coronaviruses are positive single stranded RNA viruses, and are members of Coronaviridae family. Coronaviruses localize in respiratory tract and are usually known as common cold viruses 4.At present, “severe acute respiratory syndrome coronavirus 2” or “coronavirus disease 2019” (COVI D-19) which is closely related to SARS has become a global health problem. The first-ever COVID-19 case was identified in December 2019 in Wuhan, China; however, since then the virus has spread rapidly across the world and has become a worldwide pandemic and an international concern 5. There is no evidence of vertical transmission or transmission during pregnancy 7. Incubation period of COVID-19 is estimated between 2–14 days and during this time, infected peoples are considered as asymptomatic carriers. Although infection may be asymptomatic, patients typically have fever, cough and shortness of breath. Occasionally disease progresses acutely and causes severe pneumonia, multiple organ failure and death 8. Patients with underlying medical conditions such as heart and respiratory diseases, asthma, diabetes and immunodeficiency diseases, in addition to elderly age group are high risk and more susceptible to COVID-19 infection 9. At present, there is no certain treatment or vaccination for prevention of COVID-19 and infected people are either isolated or, in critical conditions, take nonspecific or supportive care 9. Diagnosis is made based on the symptoms of the disease, chest CT (Computed tomography scan) scan and qRT-PCR (Quantitative reverse transcription polymerase chain reaction). qRT-PCR technique is the current COVID-19 (SARS-CoV-2) gold standard molecular detection method approved by CDC and World Health Organization (WHO) 11. Recently, researchers have proposed a coronavirus rapid detection method based on CRISPR/Cas system 12. CRISPR/Cas  is an adaptive immune system in archaea and bacteria against foreign genetic elements such as phages. Recently, CRISPR/Cas has become a powerful gene editing tool and a promising treatment for genetic diseases and cancers 14. In this technique, a programmable protein attaches to the target site by a guide RNA for cleavage of the target sequence. There are several types of Cas proteins that have different properties. Among them, Cas9 protein has received more attention for gene editing whereas, Cas12a and Cas13a have been more efficient in diagnosis of diseases 16. Cas12a is DNA-specific but Cas13a works with RNA which makes it convenient in detection of SARS-CoV-2. Recently, Zhang et al reported specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) technology which is a CRISPR/Cas13 based nucleic acid detection technique for rapid detection of SARS-CoV-2 18.To the best of our knowledge until March 2020, COVID-19 has been reported in 161 countries. COVID-19 is typically transmitted by respiratory droplets during sneezing and coughing 19. The test can be carried out starting with RNA purified from patient samples, as used for qRT-PCR assays, and can be read out using a dipstick in less than an hour, without requiring elaborate instrumentation 18. As a result, application of CRISPR/Cas13 based diagnosis or SHERLOCK for SARS-CoV-2 detection is much faster than detection by qRT-PCR and has high sensitivity. Consequently, SHERLOCK technology could swiftly replace qRT-PCR technique considering the high demand for rapid diagnosis tests in current global pandemic state of COVID-19.They targeted S and ORF1ab protein genes in coronavirus genome. Cas13 identifies and binds to previously determined target sequence which leads to fairly random cleavage of surrounding ssRNA molecules. SHERLOCK technology utilizes a quenched fluorescent ssRNA reporter. The presence of ssRNA corona-virus genome in samples activates Cas13 resulting in the production of quantifiable signals. Amplification of targeted DNA or RNA by Recombinase Polymerase Amplification (RPA) or reverse transcriptase-RPA (RTRPA) prior to the start of reaction improves the sensitivity of the assay. Subsequently, amplified DNA is converted to RNA by combination of RPA and T7 transcription. Ultimately, detection is made by simultaneous incorporation of the ssRNA reporter (Biotin-RNA-FITC). Viral genome is detected at attomolar concentration using SHERLOCK technology


Escherichia coli (ETEC), an essential cause of post-weaning diarrhea (PWD) in piglets, leads to significant economic losses to the pig industry. The present study aims to identify the role of ETEC total RNA in eliciting immune responses to protect animals against ETEC infection. The results showed that the total RNA isolated from pig-derived ETEC K88ac strain effectively stimulated the IL-1β secretion of porcine intestinal epithelial cells (IPEC-J2). The mouse model immunized with ETEC total RNA via intramuscular injection (IM) or oral route (OR) was used to evaluate the protective efficiency of the ETEC total RNA. The results suggested that 70 μg ETEC total RNA administered by either route significantly promoted the production of the serum IL-1β and K88ac specific immunoglobulins . Besides, the ETEC RNA administration augmented strong mucosal immunity by elevating K88ac specific IgA level in the intestinal fluid. Intramuscularly administered RNA induced a Th1/Th2 shift toward a Th2 response, while the orally administered RNA did not. The ETEC total RNA efficiently protected the animals against the ETEC challenge either by itself or as an adjuvant. The histology characterization of the small intestines also suggested the ETEC RNA administration protected the small intestinal structure against the ETEC infection. Particularly of note was that the immunity level and protective efficacy caused by ETEC RNA were dose-dependent. These findings will help understand the role of bacterial RNA in eliciting immune responses, and benefit the development of RNA-based vaccines or adjuvants.Enterotoxigenic  Escherichia coli (ETEC) is one of the main pathogens that cause post-weaning diarrhea (PWD) in piglets1. PWD is endemic and highly infectious in piglets. It typically occurs within 2 weeks after weaning and is characterized by diarrhea, dehydration, and even death3. The resulting weight loss, secondary infection, and mortality cause significant economic loss to the global pig industry. However, the most commonly applied control strategies involving antibiotics and ZnO have been restricted in many countries due to the induction of antimicrobial resistance and environmental damage6. Vaccination has become the dominant and most effective way to protect piglets against PWD. At present, antitoxin vaccines are mainly developed using virulence factors, including enterotoxin or fimbriae as antigens10. ETEC strains causing porcine diarrhea include five different fimbrial subtypes, namely K88 (F4), K99 (F5), F41, F18 and 987P (F6). Enterotoxins may be classified into heat-labile toxins (LT) and heat-stable toxins (ST)11. Due to the mixed infection of various ETEC fimbriae, the current vaccination strategies are designed to express multiple enterotoxins or fimbriae subtypes. Therefore, it will be beneficial to discover novel antigens that may improve the immunogenicity and effectiveness of the vaccines.Enterotoxigenic 13. For example, porcine reproductive and respiratory syndrome virus (PRRSV) RNA, recognized by host helicase DDX19A, stimulates IL-18 and IL-1β secretion in porcine alveolar macrophages through the activation of the NLRP3 pathway14. In addition, non-pathogenic E. coli RNA promotes the secretion of IL-18 and IL-1β in human macrophages, depending on the recognition by DHX33 and subsequent activation of NLRP3 inflammasome15. Aside from innate immunity, pathogen RNA also plays a role as an antigen to elicits adaptive immunity. When supplemented as a vaccine adjuvant, the Sendai virus-derived RNA, a RIG-I agonist, was shown to have significantly promoted the survival rate of the mice immunized with an influenza A virus vaccine against influenza A challenge16. Sander et al. discovered that the total RNA derived from non-pathogenic E. coli DH5α is necessary for heat-inactivated E. coli to trigger humoral immunity in mice17. The above studies indicated that both viral and bacterial RNA could be recognized as antigens by the PRRs of the host cells, thereby inducing both innate and adaptive immunities.Pathogen structural components such as lipopolysaccharide (LPS), DNA and RNA, are characterized as pathogen-associated molecular patterns (PAMPs). Pattern recognition receptors (PRRs) of the host cells can identify the PAMPs and trigger specific cytokine responses. As an important component of PAMPs, pathogen RNA can elicit the innate immunity in many host cells through the recognition by various PRRsE. coli RNA is capable to induce immune responses, whether or not the diarrheagenic ETEC RNA is immunostimulatory has yet to be investigated. In this study, we found that the total RNA from pig-derived ETEC K88ac strain stimulated IL-1β production of porcine intestinal epithelial cells (IPEC-J2). Therefore, we propose that the ETEC total RNA might stimulate humoral immunity and protect the animals against ETEC infection. For this purpose, BALB/c mice were administrated with the ETEC total RNA intramuscularly or orally. We examined the humoral and mucosal immunity by detecting the K88ac specific immunoglobulin levels. The protective efficiency against the ETEC challenge was also evaluated via the survival rate and weight loss. Finally, in what manner RNA immunization protected the small intestine was assessed by the histology characterization and expression levels of tight junction proteins. The results demonstrated that ETEC total RNA confers immunogenicity and effectively protects against ETEC infection in the mouse model.Although non-pathogenic The ETEC K88ac infection and the ETEC total RNA transfection significantly induced the IL-1β secretion of IPEC-J2 cells . The average serum IL-1β concentration in the 70 μg RNA intramuscularly immunized mice was slightly higher and significantly higher than that in the 50 μg and 30 μg RNA immunized mice, respectively. Thus, the serum IL-1β production was positively correlated with the immunization dosage of ETEC total RNA.To investigate whether or not the ETEC total RNA also induces the IL-1β responses in the animal model, we assessed the serum IL-1β level in the mice immunized with ETEC total RNA at 20 days post-immunization (dpi) using ELISA. As shown in Fig. P < 0.01) , while the 70 μg RNA administration caused a slightly higher IgA response than the 50 μg RNA administration  were respectively immunized with 30 μg, 50 μg, and 70 μg ETEC total RNA via IM. The vaccine administrated groups performed as a positive control, while the PBS group acted as a negative control. A recommended dosage (40 μL) and a suboptimal dosage (8 μL) of the vaccine were administered to the animals via IM, respectively. The serum immunoglobulin levels were measured at 20 dpi using ELISA. The average concentrations of ETEC k88ac specific immunoglobulins  in the ETEC RNA and the vaccine immunized mice were all significantly higher than those in the PBS treated mice (P < 0.05 and P < 0.05). The IFN-γ/IL-4 ratio in the RNA administrated group via IM was significantly lower than that from the control group (P < 0.05). It indicates the ETEC RNA influences cytokine production, resulting in a shift favoring a Th2 response. Interestingly, in animals immunized with the same amount of ETEC RNA via OR, the IFN-γ/IL-4 ratio was slightly higher than the control group. Thus, the RNA delivery route affects the Th1/Th2 cytokine balance differently. A considerably lower IL-4 level was detected in the ETEC vaccine administrated group at 20 dpi. Therefore, its IFN-γ/IL-4 ratio was significantly higher than the control group (P < 0.01), indicating the inactivated ETEC vaccine induced a stronger Th1 response.To detect whether ETEC RNA immunization affects Th1 and Th2 cytokine production, we measured the serum levels of Th1 cytokine IFN-γ and Th2 cytokine IL-4 by ELISA at 20 dpi . In order to clearly reflect the protective function of the ETEC RNA as an adjuvant, an 8-μL dose of vaccine was used to co-administrate the mice with 70 μg ETEC total RNA. A ninth group of mice was orally immunized with 70 μg ETEC total RNA to compare the effects of RNA delivery routes. At 23 dpi, the mice were challenged with 3 × 108 CFU of ETEC K88ac bacteria, and the deaths and average body weight of each group were recorded within 96 h post-challenge  were immunized with ETEC vaccine (8 μL), ETEC total RNA , vaccine/RNA mixture , PBS, ETEC total RNA treated with RNase A, and We also monitored the body weights of the mice within 96 h post-challenge Fig. B. ExceptTo investigate how ETEC RNA immunization protects the physiological structure of the murine small intestine, we observed the histopathological characteristics of the duodenums after the ETEC challenge. In comparison with the unchallenged mice, the small intestinal villi from the RNA immunized mice exhibited shorter finger-like luminal projections with an enlarged crypt region post-challenge Fig. A, B. ButTo evaluate in what manner ETEC RNA immunization affects the integrity of the small intestines following the ETEC challenge, we detected the mRNA expression of the tight junction proteins zonula occluden-1 (ZO-1) and occludin in the duodenums using qRT-PCR. After the ETEC challenge, the transcript levels of ZO-1 and occludin in the RNA immunized group declined to 53.8% and 44.4% of the unchallenged animals Fig. D. MeanwhThis study observed that both active ETEC bacteria and ETEC total RNA could stimulate the inflammation reactions of IPEC cells, thereby inducing the secretion of IL-1β. Besides, serum IL-1β and ETEC specific immunoglobulins were significantly promoted in the mouse model administrated with ETEC total RNA orally or intramuscularly. The ETEC total RNA also augmented strong mucosal immunity by elevating secretory anti-K88ac IgA in the intestinal fluid. The RNA induced humoral and mucosal immunity played a role in protecting the animals against the ETEC challenge. Furthermore, the protective efficacy of the ETEC total RNA positively correlated with its dosage. Our results proved that the ETEC total RNA exhibited immunogenicity to induce K88ac specific antibodies. Supplementing with the ETEC total RNA as an adjuvant significantly improved the protective efficacy of the inactivated ETEC vaccine.13. Many previous studies have discovered that viral and bacterial RNA could stimulate host innate immunity. For example, RNA derived from the porcine reproductive respiratory syndrome virus (PRRSV) triggers the production of IL-18 and IL-1β in porcine alveolar macrophages14. Reoviral dsRNA stimulated IL-18 and IL-1β secretion in human macrophages15. Both Mitoma et al. and Sander et al. reported that the total RNA of nonpathogenic E. coli K12 (strain DH5α) could activate the IL-1β production of human and murine macrophages through the NLRP3 inflammasome pathway17. In this study, we detected that the total RNA of a pathogenic ETEC K88ac strain stimulated the IL-1β responses in the porcine intestinal epithelial cells and a mouse model. While Loss et al. found that the ETEC (K91: K88) stimulation induced IL-8 secretion rather than IL-1β18. In our experiment, an MOI lower than 400 was not able to induce IL-1β secretion (data not shown). Therefore, the stimulation of distinct inflammatory factors might be affected by bacterial strain types or inoculation concentrations. However, the specific host PRRs that recognize ETEC RNA require further identification, and the downstream signaling pathways to activate the secretion of proinflammatory factor IL-1β also need to be characterized.Microbial RNA could be recognized by the host innate immune system through the PRRs, such as Toll‐like receptors (TLRs), retinoic acid inducible gene‐I (RIG‐I)–like receptors (RLRs), and nucleotide-binding oligomerization domain–like receptors (NLRs)16. Sander et al. discovered that live nonpathogenic E. coli significantly induced higher humoral responses than heat-killed E. coli (RNA degraded). While supplementing heat-killed E. coli with isolated E. coli RNA significantly increased serum IgG1 and IgG2b17. All the studies above revealed that RNA is a critical factor in stimulating the production of immunoglobulin subclass IgG for humoral immunity. Coherently, several studies have reported increased IgG responses to ETEC or ETEC heat-labile toxin20. In the present study, we further proved that ETEC RNA alone could induce serum specific IgG in our mouse model. Studies Sander et al. and Barbet et al. did not show any differences in serum IgM induction between mice immunized with live E. coli and those with heat-killed E. coli, thereby suggesting that E. coli RNA is not necessary for IgM induction21. However, we have detected a significant level of circulating IgM in the animals immunized with ETEC total RNA. It indicated that ETEC RNA alone is sufficient to induce IgM production. IgM is the first antibody produced during the initial response to an antigen and usually reaches peak titers within a month after exposure. Nadeau et al. and Fairbrother et al. reported significant serum anti-K88 IgA and IgM levels of the piglets vaccinated with two specific vaccines at 21 dpi, resulting in effective protection against the ETEC challenge22. Our results also suggested that ETEC total RNA administration enhanced the serum IgA and IgM productions at 20 dpi. Like the reported ETEC vaccines, the K88ac total RNA was also capable of inducing a 20-day duration of protection.Several previous studies have indicated that pathogen RNA is a potential stimulus that may trigger animal humoral immunity. Martinez-Gil et al. reported the Sendai virus RNA enhanced specific IgG responses in mice co-administrated with influenza A vaccine23. SLA-SE, the agonist of a well-characterized PRRs TLR4, was found to enhance murine intestinal IgA responses of an ETEC recombinant vaccine as an adjuvant24. Coherently, we detected a notable increase of ETEC K88 specific IgA level in the intestinal fluid of mice received ETEC total RNA via IM and OR, indicating the ETEC RNA itself delivered by either route could promote the protective immunity at mucosal surfaces. Our histological studies revealed that the murine intestinal structure was damaged after the ETEC challenge in the ETEC RNA administrated mice. Since the mice experienced weight loss right after the ETEC challenge, one possible explanation could be the bacteria colonized the intestinal mucosa and caused the damage, but the mucosal immunity finally protected the animal against the infection. However, we observed an obvious inflammation symptom at 18 h post-ETEC RNA immunization during the murine intestine dissection (data not shown). Moreover, intestinal inflammation caused by ETEC K88 infection and K88 fimbriae immunization has been reported in piglets23. Thus, we could not exclude the possibility that the intestinal damage might be due to the inflammation caused by the RNA immunization rather than the ETEC challenge.ETEC colonize the mucosal surfaces of the small intestine using their fimbrial antigens. Therefore, ETEC vaccines are developed to obtain mucosal immunities against ETEC invasion. Secretory IgA was detected in porcine intestinal tissues immunized with ETEC fimbriae vaccine25. T-cells can be divided into Th1 and Th2 cells. Th1 cells secrete cytokines such as IL-2, IFN-γ, and TNF-β to trigger cell-mediated immunity. In contrast, Th2 cells produce IL-4, IL-5, and IL-10 to evoke humoral immune responses. Since IFN-γ and IL-4 are mutually antagonistic, the IFN-γ/IL-4 ratio could represent the Th1/Th2 balance26. Delisle et al. found enhanced IFN-γ and IL-4 mRNA expression in the intestinal tissue of weaned piglets orally immunized with ETEC K88 fimbriae that adjuvanted with CpG27. In line with this, we detected elevated serum levels of both IFN-γ and IL-4 by ELISA in the mice received ETEC RNA administration. Th1/Th2 balance could be influenced by many aspects, such as antigen types, antigen dose, and cytokines28. Lee et al. reported that mice vaccinated with F4 fimbriae that orally delivered with thiolated eudragit microsphere (TEMS) triggered similar levels of IgG1 and IgG2a in mice, indicating a mixed Th1 and Th2 immune responses29. While the BALB/c mice orally immunized with a live attenuated ETEC vaccine that express fusion enterotoxins induced a Th2 type response30. Our results suggested that delivery methods affected the Th1/Th2 bias in the immune responses to the ETEC total RNA. The ETEC RNA administered by the intramuscular injection resulted in a stronger Th2 cytokine response. While the ETEC RNA administered by the oral route slightly increased the IFN-γ/IL-4 ratio compared to the control group, indicating a shift toward Th1 response.T-helper cell differentiation into distinct subtypes plays a role in mucosal immunity through maintaining the equilibrium between antigen responsiveness and tolerance16. When supplementing as an adjuvant, nonpathogenic E. coli total RNA strongly increased the immunity levels of the mice administrated with heat-inactivated E. coli17. In previous studies, TLR agonists and attenuated bacterial toxins have been widely used as ETEC vaccine adjuvants to promote protection efficacy31. Whereas the role of ETEC total RNA as an adjuvant has not been evaluated yet. For this purpose, we administrated a group of mice with 8 μL ETEC vaccine plus 70 μg ETEC RNA as an adjuvant. Due to the suboptimal protective level, the 8 μL dose of the vaccine was selected to reflect the function of the RNA adjuvant. Compared to the 12.5% survival rate in the vaccine administration group, the inclusion of ETEC RNA elevated the survival rate to 75% in the vaccine/RNA mixture immunized group. The ETEC total RNA administration either by IM or OR protected the mice against the ETEC K88 challenge with a survival rate of 75% and 87.5%, respectively. The protective efficiency of immunization with other bacterial RNA has not yet been reported. We immunized the animals with a single dose, but a booster vaccination may improve the protective efficacy. Martinez-Gil et al. reported that mice immunized with an in vitro-transcribed Sendai virus RNA as adjuvant 100% survived against an influenza virus challenge16. It reveals that in vitro synthesized RNA with the specific pathogen-origin sequence achieved high protective efficiency, thereby indicating that antigenic RNA has the potential to be manipulated and utilized in vaccine adjuvant development.The immunostimulatory activity of pathogen RNA is usually used to develop adjuvants to enhance vaccine effectiveness. For example, RNA derived from Sendai virus significantly improved the performance of an influenza A virus inactivated vaccine as an adjuvant16. The composition of viral RNA is relatively simple compared to the bacterial RNA, which is composed of various types. Interestingly, it is reported that different types of E. coli RNA trigger the host immunities differently. All three types of E. coli DH5α RNA  could stimulate IL-1β responses in human macrophages via NLRP3 inflammasome activation17. Two studies reported that only E. coli DH5α derived mRNA could activate the Nlrp3 inflammasome in murine macrophages, but tRNA and rRNAs did not32. There is no evidence it is the same case for the pathogenic ETEC RNA. But rRNA, which comprises about 80% of the total RNA, seems to critically affect the protection efficiency. In order to improve the immunostimulatory responses of the ETEC RNA, the protective effect of different ETEC RNA types either in the mouse model or in the piglets need to be further studied.We need to point out that 70 μg RNA was a large dose to achieve the desired protection. Synthetic virus RNA can produce high protective effects at a small amount when used as vaccine adjuvantsThe animal experiment in this study was approved by the Animal Ethical and Welfare Committee (AEWC) of the College of Veterinary Medicine at Hebei Agricultural University . The animals were treated in strict adherence to the recommendations in the China National Institute of Health guidelines.E. coli strain (O147: K88ac) and Salmonella strain isolated from diarrheic piglets were obtained from the China Veterinary Culture Collection Center . ETEC K88ac strain was confirmed with PCR amplification of the K88ac fimbriae gene.The IPEC-J2 cells were purchased from Guangzhou Jennio Biological Company . The enterotoxigenic The IPEC-J2 cells were infected at a multiplicity of infection (MOI) of 400 for 24 h. For the positive control, IPEC-J2 cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 12 h, followed by adding 5 mM ATP for another 5 h of incubation. The ETEC total RNA was isolated using a Bacteria RNA Extraction Kit  according to the manufacturer’s instructions. The IPEC-J2 cells were stimulated with 40 μg ETEC total RNA delivered by lipofectamine 2000  for 8 h. For the negative control, the same amount of total RNA was treated with RNase A  following manufacturer’s instructions. The cells were also treated with the same volume of lipofectamine 2000. The culture supernatants were then collected for the detection of IL-1β using Enzyme-Linked Immunosorbent Assay (ELISA) Kit for Interleukin Beta .E. coli Trivalent Vaccine for Newborn Piglets against ETEC K88, K99 and 987P strains was purchased from Shandong Huahong Biological Engineering Co. . This ETEC vaccine contains K88, K99 and 987P antigens . The 64 mice were divided randomly into eight groups (n = 8), immunized with (i) PBS, (ii) 30 μg ETEC total RNA, (iii) 50 μg ETEC total RNA, (iv) 70 μg ETEC total RNA, (v) 8 μL ETEC vaccine, (vi) 40 μL ETEC vaccine, (vii) 70 μg ETEC total RNA treated with RNase A, (viii) 70 μg Salmonella total RNA via intramuscular injection. The ETEC vaccine and the RNA were all prepared in PBS with a final volume of 100 μL. The ETEC total RNA was treated with RNase A  following the manufacturer’s instructions. A ninth group of mice (n = 8) was immunized with 70 μg ETEC total RNA via oral gavage. The whole blood samples were collected from the submandibular vein at 20 days post-immunization (dpi), respectively. The whole blood was left at room temperature for 2 h, followed by 4 °C overnight, then centrifuged at 4 °C for 20 min at 1200×g to separate the serum. The intestinal fluid was collected by flushing the small intestines with 2 mL of PBS, followed by centrifugation at 3000×g for 10 min at 4 °C to collect the supernatant. The concentration of serum IL-1β was detected using a RayBio Mouse IL-1β ELISA Kit . K88ac specific antibodies  were evaluated by ELISA using commercial kits purchased from Jiangsu Meimian Industrial Co., China. IL-4 and IFN-γ were assessed by ELISA using commercial kits purchased from USCN Life Sciences, China.The specific pathogen-free (SPF) BALB/c mice  were purchased from SPF (Beijing) Biotechnology Co., Ltd. . The Inactivated Salmonella total RNA via intramuscular injection. The ETEC vaccine and ETEC total RNA were all prepared in PBS with a final volume of 100 μL. A ninth group of mice (n = 8) was immunized with 70 μg ETEC total RNA via the oral route. At 23 dpi, all groups were challenged with 3 × 108 CFU of ETEC via intraperitoneal injection. The survival and weight loss were monitored for 96 h post-challenge. A tenth group was used as a positive control, which remained unimmunized and unchallenged. The duodenum samples of the mice from the 70 μg RNA immunized group, PBS group, and positive control group were collected at 96 h post-challenge, rapidly frozen in liquid nitrogen, and stored at − 80 °C for further experiments.For the ETEC challenge, 64 BALB/c mice were randomly divided into eight groups (n = 8), immunized with (i) 30 μg ETEC total RNA, (ii) 50 μg ETEC total RNA, (iii) 70 μg ETEC total RNA, (iv) 8 μL ETEC vaccine, (v) PBS, (vi) 8 μL ETEC vaccine plus 70 μg ETEC total RNA, (vii) 70 μg ETEC total RNA treated with RNase A, (viii) 70 μg The duodenum samples were immersed in formalin fixation solution and sent to Wuhan Cloud-Clone Co.  for embedding into paraffin blocks. The paraffin sections were sliced using a Rotary Microtome . The sliced sections were stained using a Hematoxylin–Eosin/HE Staining Kit , sealed with neutral resin, and observed under a microscope .The duodenum samples plus TRIzol reagent (3 mL/100 mg)  were ground with a glass tissue homogenizer, followed by standard procedures of RNA isolation via TRIzol reagent. The cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit , then qRT-PCR was carried out with iQ SYBR Green Supermix . The primers of occludin, ZO-1, and GAPDH used in qRT-PCR are listed in Table t-test via the IBM SPSS Statistics Version 19 (https://www.ibm.com/products/spss-statistics). The data are presented as mean ± SD for each experiment. P ≤ 0.05 was considered statistically significant. All of the experiments were performed in triplicate.Statistical analyses were performed using Student’s


Surgical treatment of spinal hepatocellular carcinoma metastasis after Liver transplantation (LT) is a clinical challenge. We herein report the clinical outcomes of the first case of a patient with T11 from hepatocellular carcinoma metastasis after systemic chemotherapy following LT combined with mesenteric resection and colectomy, who was successfully treated with En Bloc spondylectomy.The patient with HCC was a 40-year-old man, who had received LT combined with mesenteric resection and colectomy 15 months before. His main symptom was progressive back pain because of T11 metastasis. PET examinations showed a solitary metastasis at T11 without recurrence in the liver and metastasis in the other organs.The patient was diagnosed with the T11 vertebra HCC metastasis after LT combined with resection of HCC mesenteric metastasis and colon metastasis.Five cycles of systemic chemotherapy following LT were performed for preventing HCC metastases. However, the right abdominal wall metastasis was found 9 months after LT, followed by T11 metastases thereafter. Immediate resection of the right abdominal wall metastasis was achieved. En Bloc spondylectomy of T11 vertebra was chosen as a treatment for metastasis to T11. After T11 surgery, the patient showed obvious pain relief. However, At 3 months after T11 surgery, a grafted liver metastasis and multiple nodules metastasis in the greater omentum region were revealed with CT imaging, At 5 months after T11 surgery, multiple lung metastases were discovered by MRI. The patient was performed 5 cycles of chemotherapy, 3 times of infusion of iodine [131I] meximab and 3 times of TACE after T11 surgery. Multiple bone metastases were treated with radiotherapy.The patient died 29 months after LT combined with mesenteric resection and colectomy because of recurrence in the liver and metastasis in the lung.En Bloc spondylectomy may be a therapeutic choice for patients with progression after systemic chemotherapy for the solitary spinal metastases after LT combined with mesenteric resection and colectomy, which has a survival benefit without local recurrence at the surgical site. immunosuppressant after LT may result in worse immune function, which leads to HCC more prone to recurrence and bone metastasis. In the past, the presence of BMs associated with HCC was considered rare and the outcomes were typically poor. However, with recent advances in radiographic imaging methods and treatment modalities, BMs in patients with HCC are more commonly detected. Liver transplantation (LT) is the curative therapy for early HCC, but LT for HCC maintains a risk of HCC metastasis and recurrence after LT. Few reports are available exploring operative approaches for bone metastases after LT secondary to HCC. In this report, we present our experience of operative approaches for bone metastases and recurrence after LT combined with mesentery and colectomy management in this case.Approximately 20% of bone metastases (BMs) are caused by extrahepatic metastasis.2,5 The patient underwent an En Bloc spondylectomy of T11 vertebra metastases using a posterior approach. The pedicle screws were inserted into both sides of the T9, T10, T12, and L1 vertebrae through the pedicle, and then the titanium alloy rods were fixed on the screws. Reconstruction was achieved by using a artificial vertebral body at the original T11 position after the T11 En Bloc spondylectomy  in LT (16 cases) and non-LT patients (27 cases) was 20 and 11 months, respectively. This did not mean that the median survival time of LT patients is longer than that of non-LT patients. The operative procedure is considered to be a factor determining survival, so patients who develop a recurrence after transplantation have higher life expectancy if they undergo surgery.The majority of patients with recurrences after LT have multiple sites of disease spanning different therapeutic fields. Surgeries for spinal metastases are effective for removing masses, while decompression with resection helps to improve pain and alleviate neurological blockade. The walking condition of patients coupled with radiation therapy after surgery was significantly improved compared with patients undergoing radiation alone despite some reports contradicting that with the results a study conducted by Rades and colleagues.–12 bone is the third largest extrahepatic metastasis site after grafted liver and lung (18–33%). Subsequent studies identified several clinical characteristics and prognostic factors of bone metastasis after LT for HCC. The most frequently affected site in these patients was the thoracic vertebrae, accounting for 15.1% of all patients. The median survival time in this group was 8.6 months, and liver failure or tumor progression was the most common cause of death. In our case, the patient was found to have metastasized to the grafted liver, lung, and bone. Despite the active treatment of spinal metastasis, additional metastases on the grafted liver and lungs still exist, resulting in progressive decompensation of liver function and respiratory failure caused by lung metastasis. The patient eventually died of the disease 29 months after LT. Among the affected sites, the bone metastasis responded poorly to irradiation, with the bone lesion continuing to grow larger and more over time.Studies have shown that in patients receiving LT for HCC,The patient developed bone metastasis 15 months after systemic chemotherapy following LT combined with mesenteric resection and colectomy, suggesting that the patient has two possibilities. The first possibility is bone metastasis during LT, which was simply unable to be detected by current diagnostic methods. The second case is that immunosuppression after LT leads to immune insufficiency, which will make HCC more prone to recurrence and bone metastasis. In this study, we prefer the latter interpretation because the first bone metastasis in our patient was found after 15 months of immunosuppression therapy following LT rather than immediately after LT. Patients with LT usually receive immunosuppressive therapy after LT,,15 which can accelerate tumor recurrence and metastasis. At present, there is no reliable conclusion that any specific immunosuppressants have an effect on the recurrence of HCC; however, compared with other factors, immunosuppressive load seems to play a decisive role in the recurrence of HCC.,17 The original immune state of patients can increase the survival rate of patient and graft,–20 the current trend is to take into account the original immune state of patient, as far as possible to reduce the immunosuppressive load. This type of treatment usually requires appropriate adjuvant chemotherapy to minimize immunosuppression.The patient's burden of disease including the immune status was early detected and assessed by the LT team from the time of transplantation in our hospital. Studies have shown that post-transplant recurrence is more severe than that post-hepatectomy because the immunosuppressive drugs used after transplantation have many side effects on the overall immunity of the patient.When long-term survival is expected, posterior decompression for symptomatic spinal metastasis is very common, as well as total En Bloc spondylectomy for a single metastatic site.–25 When we review the literature, only two case reports,27 have investigated the application of En Bloc spondylectomy in the treatment of HCC solitary spinal metastasis after LT.Several studies have reported that En Bloc spondylectomy has a survival benefit in patients with solitary spinal metastasis derived from different tumors. reported that the preceding radiotherapy along for solitary T12 vertebra metastasis after LT in a case report was unable to control tumor progression. Accordingly, the patient was treated via En Bloc spondylectomy of T12. After surgery of T12, the patient developed an infection of the spinal incision, which was thought to be related to radiotherapy. Based on the patient's medical condition, the patient was predicted to have a very short survival and a poor overall quality of life; however, the patient ultimately lived beyond the predicted survival time of 6- to 12-month and no recurrence at the T12 surgical site was noted at a follow-up postoperatively.Hu et al reported that a case underwent radiation therapy for solitary T7 vertebra metastases after LT, but the paralysis in the lower legs aggravated further, which was thought to have been the result of spinal cord injury caused by radiotherapy. Accordingly, the patient was treated via En Bloc spondylectomy of T7 through a posterior approach. The patient was satisfied with the treatment without sign of recurrence at the T7 site of the operation at the 1.5-year follow-up.Hiroaki et al and injure the spinal cord, and then they was treated via En Bloc spondylectomy for solitary spinal metastasis from HCC after LT.From these only two case reports concerning En Bloc spondylectomy for solitary spinal metastasis from HCC after LT, two patients all received radiotherapy first, but radiotherapy could not control tumor progression and damaging spinal cord, while En Bloc spondylectomy can theoretically completely remove the tumor and reduce the risk of local recurrence. This patient had appropriate liver function without HCC recurrence in the right abdominal wall after resection of right abdominal wall metastasis following LT and no metastases except the solitary T11 metastasis. Therefore, we performed En Bloc spondylectomy, and expected for long-term survival and long-term local control. The patient ultimately survived beyond the predicted time of 6- to 12-month and no recurrence at the T11 surgical site was seen at the 14-month follow-up postoperatively.We also considered radiotherapy before En Bloc spondylectomy for solitary T11 metastasis after LT, but radiotherapy has the risk of losing control of tumor development,27 concerning En Bloc spondylectomy for solitary spinal metastasis from HCC after LT.The case in our study showed En Bloc spondylectomy had a survival benefit without local recurrence at the T11 surgical site in the treatment of HCC solitary spinal metastasis after LT combined with mesenteric resection and colectomy, which is consistent with the results of previous only two case reports,27 our patient receiving LT combined with mesenteric resection and colectomy were typically diagnosed as advanced HCC, with poorly controlled intrahepatic tumors that extend to surrounding mesenteric and colonic soft tissue. In our patient due to the mesentery and colon invasion by the tumor, a mesenteric resection and colectomy was performed during LT. When we review the literature, we could not find any other HCC in liver case with a mesentery and colon invasion which was treated by LT combined with mesenteric resection and colectomy. We did not preserve the mesentery and colon due to it being full of metastasis in the mesentery and colon. It is unclear yet, whether a mesentery and colon preserving surgery of LT is suitable or safe for this visceral organs HCC metastasis.Compared with only receiving LT patients in previous only two case reports, The patient here had good coagulopathy and liver function without surgical contraindications at the time of discovery of the bony metastases and the first bone metastasis surgery.Taking into consideration that the prediction of patients with HCC with spinal metastasis is poor, a healthy liver and good liver function after LT may enable a successful surgery. The case presented here suggests that total En Bloc spondylectomy may be a feasible treatment option for patients with solitary spinal metastases after LT combined with mesenteric resection and colectomy, which had a survival benefit without the surgical site recurrence.To the best of our knowledge, no reports have been published about En Bloc spondylectomy for solitary spinal metastasis of HCC after LT combined with surgery of other visceral organs for HCC. Based on our experience, we suggest that only patients with solitary spinal metastases and appropriate liver function after LT combined with surgery for metastasis of other visceral organs consider any surgical intervention, which is consistent with the results of previous researchers.Conceptualization: Jingen Hu, Caibao Hu.Data curation: Jingen Hu.Formal analysis: Jingen Hu.Investigation: Jingen Hu.Resources: Jingen Hu.Supervision: Jingen Hu.Validation: Jingen Hu.Writing – original draft: Jingen Hu.Writing – review & editing: Caibao Hu.


C. elegans. In the original design, the floxed unc-119+ selectable marker was incorporated into the tag. For site-directed mutagenesis, the selectable marker is useful but a tag is unnecessary. We have developed a new set of vectors dedicated for introducing point mutations using unc-119+ selection. These new vectors contain only a floxed unc-119+ selectable marker in the reverse orientation of a synthetic intron (‘syntron’) that lacks both 5’ and 3’ splice sites. This unc-119+ module can be inserted directly into a native intron. Alternatively, splice site sequences can be appended for insertion into a coding sequence to generate a syntron in the coding sequence. The desired point mutations are designed into the homology arms of the selection construct. SapTrap is used to assemble a plasmid encoding the guide RNA and the template. After injection and selection of unc-119+ animals, the selectable marker can be removed by expression of Cre recombinase to generate an animal with only the desired point mutation and a single Lox site in the gene of interest.SapTrap assembly generates CRISPR targeting vectors using a Golden Gate cloning strategy . The original toolkit was designed for inserting tags, such as fluorescent proteins or affinity tags, into protein coding genes in Reagents for point mutation vectors differ from reagents for tagging vectors in three ways:(1) Different cohesive-end sequences are used to join the homology arms to the marker module.(2) Splice-site sequences must be added to the homology arms if inserting into a coding region.unc-119+ cassette. If the point mutation were placed beyond the unc-119+ cassette, then repair could drop off of from the repair template before encountering the mutation, thereby failing to fix the mutation in the genome. Then some Unc-119+ animals would lack the mutation, resulting in significant numbers of false-positive strains.(3) The point mutation, the guide RNA cut site, and selectable marker must be carefully arranged. Specifically, point mutations must be positioned between the cut site and the selectable marker. This arrangement ensures that all Unc-119+ animals have incorporated the desired point mutations. Homology-directed repair fixes double stranded breaks by copying the repair template DNA across the break. The extent of copying beyond the immediate vicinity of the break is stochastic. By selecting for Unc-119+ incorporation, we select for repair events that copy the repair template between the break and the We recently released the SapTrap Builder software, a desktop utility for designing reagents for SapTrap assemblies . To generate reagents for introducing point mutations using the new selectable marker-only vectors described here, simply select ‘Native Intron’ or ‘Syntron’ in the ‘Tag Type’ field during design. SapTrap Builder will add the correct cohesive ends to the homology arms, add splice-site sequences when needed, and monitor for the arrangement of the cut site, marker insertion site and mutations, alerting the user to unfavorable arrangements.Plasmids are available through Addgene.