Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-‘omics approach.Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-‘omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV  The metabolome is an incredibly diverse collection of small molecules  in biological systems involved in virtually every cellular process, including cellular energy production, macromolecule synthesis, epigenetic modifications, cell signalling and more (for recent reviews see ). It resWhile metabolomic profiling is useful in identifying metabolites or metabolic pathways involved in a phenotype, it fails to capture metabolic dynamics or directionality and can be difficult to interpret in isolation . Conversversus a traditionally extracted control. As such, it is unknown whether this additional processing influences the downstream ‘omics readout and ultimately the biological interpretation.Both RNAseq and mass-spectrometry-based metabolomics rely on the destruction of the sample from which the target molecules are isolated. The sample must be regenerated or sampled multiple times for additional analyses, introducing sampling errors and limiting feasibility in low-quantity or rare sample types. For metabolomics, the wide range of compound chemistries present in the metabolome poses a particular challenge as no extraction method is suitable for the entire metabolome. A widely used approach to address this problem is the homogenization of the sample with ice-cold 80% methanol, which both quenches metabolism through protein precipitation and extracts polar and semi-polar metabolites involved in central carbon metabolism ,30. Afteversus non-infected controls) is an excellent model system that features strong metabolic and transcriptional responses, providing rich biological contrast, and that is large enough to be sampled multiple times to allow for direct comparison of extraction methods. Our results demonstrate that prior metabolite extraction does not impact RNAseq quality. We further use integrated single-sample metabolomics and transcriptomics data that support a novel role for pyrimidine metabolism in the hepatic response to LCMV infection.To address this gap, the main objective of this study was to determine the effects of prior metabolite extraction on RNAseq using liver tissue from mice infected with a model of acute lymphocytic choriomeningitis virus (LCMV) infection. The liver is both a master regulator of systemic metabolic homoeostasis and an immunological hub owing to its large population of non-parenchymal cells including phagocytes  and lymphocytes . LCMV iad libitum access to water and a standard chow diet. At 11 weeks of age, mice were randomly selected to receive an intraperitoneal injection of lymphocytic choriomeningitis virus (LCMV)  or PBS vehicle (Veh) [2 for later processing. All animal protocols were approved by the Van Andel Institute’s Institutional Animal Care and Use Committee.Twenty male-C57/BL6 mice were selected for the study. All mice were maintained in a climate-controlled environment on a 12 h:12 h light–dark cycle with le (Veh) . 2.5 day2. Being careful to avoid thawing of powdered liver, two roughly 30 mg aliquots of each liver were rapidly weighed into liquid N2 chilled tubes. An aliquot was weighed into a liquid N2 chilled 1.5 mL Eppendorf tube for later RNA extraction. The other aliquot was weighed in to a 2.0 mL Omni Bead Ruptor  tube for metabolite extraction. Precise weights were then recorded, and samples were stored at −80°C until processing.Frozen livers were pulverized into a fine powder using a mortar and pestle pre-chilled in liquid N2. Aliquots of 30–40 mg were prepared by weighing rapidly to avoid thawing. Samples were homogenized in a bead mill homogenizer with a liquid N2 chilled homogenization chamber to prevent sample warming. Extraction solvent volume was adjusted for each sample to achieve a fixed 40 mg tissue/mL extraction solvent ratio. For plasma, 20 µL of plasma was extracted in 980 µL of 80% MeOH (v/v). Both liver and plasma samples were incubated on wet ice for 60 min to ensure complete metabolite extraction and precipitation of protein and nucleic acids. Extracts were then centrifuged at 17,000 × g for 10 min at 4°C. The supernatant was transferred to a fresh 1.5 mL Eppendorf tube, dried in a Speedvac (Genevac) and then stored at −80°C for later LC/MS analysis. The insoluble pellet from liver extracts was dried and stored at −80°C for subsequent RNA extraction.Metabolites from the liver and plasma were extracted with 80% methanol (v/v). For liver, frozen tissue was pulverized with a mortar and pestle under liquid N2O), and mobile phase B was 100% methanol. Both mobile phases contained 10 mM of the ion-pairing agent tributylamine , 15 mM acetic acid and 2.5 µM medronic acid . The LC gradient was as follows: 0–2.5 min 100% A, 2.5–7.5 min ramp to 80% A, 7.5–13 min ramp to 55% A, 13–20 min ramp to 99% B, 20–24 min hold at 99% B. Flow rate was 0.25 mL/min, and the column compartment was heated to 35°C. The column was then backflushed with 100% acetonitrile for 4 min (ramp from 0.25 to 0.8 mL/min in 1.5 min) and re-equilibrated with mobile phase A for 5 min at 0.4 mL/min. Full LC-MS parameters and measured metabolite peak areas are available in Supplemental File 1.Metabolite profiling analysis was completed using ion-paired reversed phase liquid chromatography using an HPLC  coupled to a triple quadrupole mass spectrometer  with electrospray ionization operated in negative mode. The column was a ZORBAX Rapid Resolution HD . Mobile phase A was 3% methanol (in Hhttps://bioconductor.org/packages/release/bioc/html/DEP.html). To start, metabolites with zero variance were filtered out, and then the remaining metabolites were assessed for missingness. Metabolites with missing data were determined to be not-missing-at-random as these metabolites had lower overall mean abundance compared to metabolites without any missingness. To address this potential source of bias, metabolites with less than 30% missingness were imputed from a left truncated distribution with parameters estimated using quantile regression (https://cran.rstudio.com/web/packages/imputeLCMD/index.html). Metabolite data were then log2 transformed, and a variance stabilizing normalization was applied to prepare data for analysis via LIMMA ebayes; both the Limma linear regression and empirical Bayes were fit using robust methods to prevent individual animals and/or metabolites from exerting excessive influence on estimates (https://bioconductor.org/packages/release/bioc/html/limma.html). Batch was included as covariate in the LIMMA linear models. Hypothesis tests were then multiple-testing adjusted via Benjamini-Hochberg to maintain a 5% false discovery rate (FDR).Differential abundance of metabolites collected from liver tissue and blood serum was analysed separately using R v 4.1.0 via a workflow, inspired by the R package DEP (https://cran.r-project.org/web/packages/betareg/index.html).RNA was isolated directly from frozen liver tissue (RNA) or from post-metabolite extracted liver tissue (MetRNA). Samples were pulverized using QIAzol Lysis Reagent and a Tissue Lyser II. RNA was then purified from the lysates using RNeasy Plus Universal Mini Kit column-based isolation per manufacturer’s instructions. Isolated RNA was quantified by Promega Quantus and measured for integrity using Agilent Bioanalyzer. We used the calculated RNA Integrity number (RIN), which used the entire electrophoretic trace of RNA sample (including degradation products) to compare the RNA integrity between our samples. Differences in unique mapping rates between LCMV vs Veh and MetRNA vs RNA were tested using beta regression via the R package ‘betareg’ . RNA libraries were then normalized, pooled and sequenced on Illumina NovaSeq S1 flow cell using paired end, 50 base pair reads to an average depth of 50 M reads per sample. Base calling was performed by Illumina RTA3, and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). These trimmed reads were then aligned to the GRCm38 reference genome using the STAR aligner v2.7 (https://pubmed.ncbi.nlm.nih.gov/23104886/). Aligned read counts were imported into edgeR (https://doi.org/10.1093/bioinformatics/btp616) wherein library sizes were normalized using the trimmed mean of M values method and pairwise comparisons for differential expression were performed using the quasi-likelihood approach as in . Raw and processed data are available under the GEO Accession ID GSE226930.Adapters and low-quality bases (Phred >30) were trimmed from raw reads using Trim_Galore (n = 10/group) enabled an expected 95% power to detect all true fold-changes >1.5 and >99% power for all fold-changes >1.75 at a read depth of 40 M and a conservative true null rate of 99.9%.The current experimental design (https://cran.r-project.org/web/packages/Hmisc/index.html). The mean absolute difference was chosen as a measure of dispersion because it is non-parametric and has a natural interpretation. The distributions of the fold-change in dispersion were then assessed for skewness, which could indicate a biased change in dispersion related to the extraction method, particularly if this bias is conserved over both LCMV and vehicle groups. Finally, the mean absolute differences from the two extraction methods were correlated using Pearson’s rho.To determine if isolating RNA and metabolites from the same sample altered variance profiles within the RNAseq gene expression estimates , we compared the mean absolute difference between samples where only RNA was isolated and samples where RNA was isolated post-metabolite extraction, stratifying them by experimental group (LCMV/Veh) . All differentially expressed genes, along with their fold-changes, were entered as gene symbols, and all differentially abundant metabolites were entered as their compound names. Metabolite fold-changes were not included to avoid the enrichment being dominated by large fold-changes as small metabolic differences can have large impacts. Pathway impact and Benjamini-Hochberg FDR adjusted significance were estimated using hypergeometric tests and MetaboAnalyst’s ‘tight integration’ method [Multi-omic pathway enrichments were performed using MetaboAnalyst’s v5.0-‘joint-pathway analysis’ where we investigated which metabolic pathways were most impacted by LCMV infection . MetRNA had an increased 260/230 ratio , which could be an indication of removal of contaminants, and improved RNA yield . Thus, the metabolite extraction carried out prior to RNA purification does not compromise RNA quality and might serve to improve contaminate removal and RNA yield.RNAseq was performed on both RNA and MetRNA samples. Unique mapping rates, i.e. the number of sequenced fragments that map to a unique location in the genome, averaged 75% for vehicle and 82% for LCMV-treated samples . The increased unique mapping rate in LCMV over vehicle was hypothesized to be due to gene activation in response to viral infection, as LCMV had more unique gene features after trimming . We further observed a small but statistically significant increase in the unique mapping rate of MetRNA vs RNA, which correlates to the improved 260/230 ratio. No additional genes were detected as a result of this small difference in unique mapping rate. Samples were diverse and expressed 15,535 unique gene features after trimming . Within individual animals, absolute transcript abundance was tightly correlated between MetRNA and RNA . Gene expression within the RNA samples explained >98% of variance in the MetRNA samples for each animal. Furthermore, after trimming, there was ~ 96% overlap in genes detected in each sample between the two extraction methods . There were no genes specific to only one extraction method, that is, if a gene had measured counts in one extraction method it was also measured in at least a subset of animals in the other.Rpph1 and Moxd1 , whereas Moxd1 had no evidence of differing between methods .Principal component analysis revealed a robust separation between LCMV and Veh groups in principal component 1 , irrespective of the extraction method, followed by intragroup differences in PC2 . The median fold-change between genes uniquely significant to an extraction method was 1.04 with 94% of the genes having a log2 (fold change) less than ±1. The probability of obtaining this amount of overlap (>85% shared DEGs) by chance, as estimated via a hypergeometric test, is <0.0001. Further, as seen in p-values were <0.001; Having demonstrated that prior metabolite extraction does not affect the distribution of differentially expressed genes, we next sought to better understand what effects extraction method has on the global dispersion of RNA. That is, does RNA extraction modality introduce or reduce noise? Within both LCMV and vehicle-injected animals, the absolute mean difference was significantly correlated between MetRNA– and RNA-only transcripts . This revealed JAK-STAT signalling pathway, linoleic acid metabolism, beta-Alanine metabolism, neutrophil degranulation, signalling by interleukins and cytokine signalling in the immune system as some of the most impacted pathways. Interestingly, there was little overlap between RNAseq metabolic pathways and those pathways identified with metabolomics data.de novo pyrimidine synthesis , nucleosides  and nucleobases  were increased (de novo nucleotide production is elevated to support increased RNA/DNA synthesis or whether nucleotides are being catabolized to produce nucleosides/nucleobases) was unclear.Finally, we integrated metabolite and RNA data and performed a combined pathway analysis ncreased . When coNt5c3) had elevated expression . The concordance of these measures provides confidence that infection with LCMV results in a strong involvement in pyrimidine metabolism.As pyrimidine metabolism was the most significantly impacted pathway in our multi-‘omic analysis, we used it as an example to highlight the potential of multi-‘omic integration. However, it is important to note that other metabolic pathways were also significantly affected. Joint-integration methods revealed other candidate pathways to explore, notably ‘Starch and Sucrose Metabolism’ and ‘Degradation of Ketone Bodies’, which were only identified as being highly impacted in the joint analysis.A general limitation of metabolomics is incomplete coverage of the metabolome. While RNAseq data can provide a comprehensive view of transcripts above a limit of detection threshold, metabolomics coverage is dependent on both analytical methods  and biological abundance. Therefore, it is important to consider that metabolomics studies, and subsequent multi-omics integration, will be biased towards the metabolites that are detectable in the method. In this light, metabolomics can be used positively, for example, to provide orthogonal validation of RNAseq results or highlight altered metabolic pathways independent of transcription. However, the use of metabolomics data to rule out certain metabolic pathways is not recommended because metabolic pathways are often incompletely covered or simply not detected. By extension, RNAseq results may be used for direct follow-up metabolomics analyses that target specific pathways/compounds of interest.Rpph1 was the only transcript affected by the extraction method. Rpph1 is a highly conserved long non-coding RNA (lncRNA) that encodes the RNA component of RNaseP. In eukaryotes, RNaseP exists as a riboenzyme with up to 10 protein subunits [Rpph1 does appear in other mRNA-sequencing datasets [Rpph1 expression in metRNA may indicate that the additional steps prior to RNA extraction improved the extraction of protein-complexed RNAs. Whether metabolite extraction similarly boosts the recovery of other lncRNAs was not evaluated here, but our data encourage further investigations in this area. Secondly, liver tissue was used as a model system due to its size (to enable multiple sampling) and its strong phenotypic response to LCMV infection. It is unknown whether RNA in tissues with different compositions, for example, high lipid content in adipose tissue, will respond the same. Finally, the tissue-to-solvent ratio was carefully controlled to avoid the potentially confounding influence of extraction efficiency, and only a single commonly used metabolite extraction modality (80% methanol) was used. These factors should be considered when designing a study to co-extract metabolites and RNA.The current study presents an in-depth evaluation of the effects of prior metabolite extraction on mRNA quality, transcriptomic coverage and phenotypic insight. However, there are limitations to our approach that warrant discussion. First, whether extraction modality affects non-protein coding RNAs was not assessed. Our analysis revealed that subunits ,48. Althdatasets . In the Often biological material is limited, and the ability to maximize information gained from the same sample is beneficial. Even when tissue is in abundance, regional heterogeneity within a tissue can limit the utility of multi-omics approaches. Our study demonstrates that sequentially extracting metabolites and RNA for metabolomics and transcriptomic analyses does not fundamentally impact gene transcript abundance. We also show that by jointly integrating changes to both omics’ datasets through enrichment analyses, we can identify metabolic phenotypes with higher statistical confidence, while gaining additional biological insight into how pathways are altered. Finally, through the implementation of these methods, we reveal a novel finding that infecting mice with LCMV results in significant and dramatic changes to pyrimidine metabolism. This work provides validation that sequential extraction of metabolites and RNA from a single sample can be confidently used for multi-omics analysis.Click here for additional data file.


Glioblastoma (GBM) comprises 45.6% of all primary malignant brain cancers and is one of the most common and aggressive intracranial tumors in adults. Intratumoral heterogeneity with a wide range of proteomic, genetic, and epigenetic dysregulation contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. To date, numerous clinical trials have been developed to target the proteome and epigenome of high-grade gliomas with promising results. However, studying RNA modifications, or RNA epitranscriptomics, is a new frontier within neuro-oncology. RNA epitranscriptomics was discovered in the 1970s, but in the last decade, the extent of modification of mRNA and various non-coding RNAs has emerged and been implicated in transposable element activation and many other oncogenic processes within the tumor microenvironment. This review provides background information and discusses the therapeutic potential of agents modulating epitranscriptomics in high-grade gliomas. A particular emphasis will be placed on how combination therapies that include immune agents targeting hERV-mediated viral mimicry could improve the treatment of GBM. In vivo studies link epitranscriptomic dysregulation with glioma pathogenesis.TCGA shows elevated regulator expression is associated with increased mortality in GBM patients.RNA modifications negatively correlate with GBM immunogenicity and thus reduce the efficacy of immunotherapies in GBM patients.RNA modification regulators represent promising therapeutic agents for reducing mortality and immunotherapy adjuvants for enhancing tumor immunogenicity.  Glioblastoma (GBM) comprises 45.6% of all primary malignant brain cancers and is one of the most common and aggressive intracranial tumors in adults . Due to GBM is a highly heterogeneous neoplasm with a wide range of proteomic, genetic, and epigenetic dysregulation . Many enFew dedicated studies of posttranscriptional modifications and their potential as therapeutic targets occurred initially. Still, in the past few years, this field has become an active field of investigation for brain tumors . SeveralA common consequence of pretranscriptional  and posttranscriptional  dysregulation is the activation of transposable elements (TEs). Interestingly, in the last decade of cancer research, a form of TEs known as endogenous retroviruses (ERVs) has been shown to play a dichotomous role in driving oncogenesis and serving as potential antigens for immunotherapy . First dRecent publications extensively reviewed how RNA modifications participate in cancer and glioma development/progression, regulate the tumor microenvironment (TME), and encourage the development of drug resistance , 9. ThisIn the context of glioma pathogenesis and progression, the most relevant RNA modifications are N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), hydroxymethylcytidine (hm5C), pseudouridine (Ψ), and adenosine-to-inosine (A-to-I) RNA editing . ElevateThe following subsections will review the physiologic role of each RNA modification on gene regulators. Furthermore, Table Methylation of the adenosine at the N6 position (m6A) is the most common modification of mRNA and is primarily found in the 3’ untranslated region (UTR) . The wriThe physiologic function of m6A modifications involves pre-mRNA splicing, mature mRNA transport, and translation , 16, 17.Adenosine methylation at the N1 position (m1A) is frequently found in the 5’ UTR of mRNA and tRNA. The tRNA methyltransferase 6/61A (TRMT6/61A) and tRNA methyltransferase 10C (TRMT10C) writers and YTHDF1/2/3 and YTHDC1 readers function to influence mRNA structural stability and increase translation , 22. AddIn addition to being found on DNA, the methylation of cytosine at the N5 position (m5C) is frequently found in the UTRs of mRNA . WritersIsomerization of uracil (U) yields the pseudouridine (Ψ) modification. This is thought to be the most abundant RNA modification, mainly accumulating in rRNAs and tRNAs . An RNA-Nucleotide base editing of adenosine to inosine is catalyzed by adenosine deaminases (ADARs) . In addiThe role of RNA modification in cancers is incredibly complex and challenging to study, primarily due to limitations in sequencing technologies. Conventional sequencing techniques such as bulk RNA-seq and scRNA-seq quantify and normalize for mRNA expression but are not specifically developed to detect RNA modifications. Taguchi et al. recently reviewed developments in epitranscriptome spatial detection and data analysis . AdvanceRNA modifications, namely m6A methylations, have been found to influence the tumor microenvironment (TME), immune cell-infiltrating characteristics, and, ultimately, patient outcomes , 42. RNAMolecular alterations characterizations such as isocitrate dehydrogenase IDH1/2 mutations, 1p/19q codeletion, and MGMT promoter methylation, have improved the accuracy of diagnostics, prognostics, and prediction of treatment response for glioma patients. However, we have yet to observe any tangible improvement in either clinical management or patient outcomes. A potential utility of epitranscriptomics is to be used in conjunction with known glioma sub-entities to further advance tumor stratification and help guide treatment selection . AccordiA critical limiting factor of most epitranscriptomic cancer studies has been the scientific indifference towards RNA modifications other than m6A. Given the integrated process of interactions between different RNA methylation modifications, studying a single type of RNA modification in tumors may not comprehensively elucidate epitranscriptomic effects. A recent study by Li and colleagues recognized this. It developed a GBM score’ based on the differentially expressed genes (DEGs) between groups showing RNA modification patterns of writers that catalyze m1A methylation, m6A methylation, APA, and A-to-I RNA editing . Out of The immunosuppressive tumor microenvironment has been identified as the primary reason for the failure of immune therapy in GBM patients; currently, there are no FDA-approved immunotherapies for GBM . AccumulEpitranscriptomic sequencing technologies may be divided into four fundamental approaches: antibody-based, reverse-transcription signature-dependent, enzyme-dependent, and chemically assisted . AntibodNovel sequencing technologies that allow simultaneous detection of multiple modifications are vital to understand the crosstalk between modifications further and drive the field of epitranscriptomics. Third-generation methods such as SMRT and Nanopore sequencing hold promise but have faced challenges, including limited signal-to-noise ratios, complex algorithms, and high error rates and costs . In addiAs occurs in pluripotent embryos, many cancers are known to reactivate transposable elements (TE) through chromatin remodeling, DNA hypomethylation, and histone modifications . TEs areGag (group-specific antigen)-Pro (protease)-Pol (polymerase) polyprotein-coding sequence. Gag is cleaved by Pro to generate a virus-like particle that contains the fusion protein and ERV mRNA. hERVs may also contain a remnant envelope (Env) and other accessory genes but are generally not infectious [Gag, Pol, and Env, which can be read and translated into functional retroviral proteins [The genome of an intact hERV provirus comprises at least 5’ and 3’ long terminal repeats (LTRs) flanking an internal fectious . HERV-K proteins .While necessary for mammalian embryologic development, ERV activation and transposition can compromise host health secondary to aberrant transcriptional regulation; thus, constant surveillance is needed to maintain homeostasis . AlthougERV regulatory networks fundamentally consist of pre-transcriptional and post-transcriptional regulatory networks . Pre-trahERVs may drive oncogenesis in two main ways: (1) indirect transcriptional regulation of oncogenes/tumor suppressors and (2) expression of oncogenic HERV proteins .There is accumulating support for hERVs as an oncogenic driver and emerging target for treatment via epigenetic silencing. On the other hand, the role of post-transcriptional silencing and RNA modifications of hERV transcripts in glioma remains virtually unexplored. This subsection briefly recounts major hERV-mediated oncogenic findings and provides context for the onco-exaptation discussion later in the review.Akin to its role in development, hERVs may serve as alternative promoters for proximal genes in malignant cells and cryptic transcription start sites to produce aberrant CDS mRNA . SimilarEnv and accessory proteins Rec and Np9 have been linked to tumorigenesis in various cancers [Through c-MYC proto-oncogene activation, HERV-K  cancers . Rec and cancers . Np9 has cancers . Interes cancers . TherefoFinally, hERVs have been proposed to be responsible for the stem-cell phenotype in cancer stem cells (CSCs) , 61. CD1Chemical modifications on DNA, RNA, and proteins  impact gene regulation. As discussed above, installing an RNA m6A modification alters mRNA stability and translation. Furthermore, emerging data suggests that m6A methylation influences physiological regulation beyond post-transcriptional mechanisms . While lA vital function of heterochromatin is restraining the activity of embedded satellite repeats and transposable elements . EndogenThe current explanation for these findings is that m6A RNA modifications catalyzed by Mettl3 are known to interact with Ythdc1 readers. This interaction, confirmed via chromatin immunoprecipitation followed by sequencing (ChIP-seq) with enrichment at H3K9me3-rich transposable elements, has a role in mediating retrotransposon silencing and maintaining mESC identity . In addiDespite some inconsistencies in the proposed mechanism, these studies –79 proviIt is currently unknown if and how RNA modifications other than m6A feedback on epigenetic checkpoints.Viral mimicry describes an active anti-viral cellular state triggered by an endogenous stimulus. It may evoke innate and adaptive immune responses and can be triggered by cytosolic RNA or DNA .Two landmark papers in 2015 describe “viral mimicry” as a process involving inhibition of epigenetic silencing, retrotransposon transcription, and IFN activation. The host cells interpret aberrant repetitive element RNA expression as a viral infection and activate an IFN response , 83. RecIn glioblastoma cell lines, DNA methyltransferase inhibition (DNMTi) increased the expression of TE and HERV-derived peptides . HoweverK27M mutation in high-grade gliomas [K27M gliomas do not have elevated IFN signaling, likely secondary to a cancer-specific compensatory mechanism. Treatment of high-grade glioma with either DNMTi or HDACi has been shown to more strongly promote MAVS-dependent induction of IFN and IFN-stimulated genes in H3.3K27M cancers than H3.3 wild-type cancers. The resulting enhanced dsRNA responses promote PKR-mediated cell death [Several recurrent cancer-driving mutations are known to activate TEs that prime tumors for viral mimicry induction. One of the most studied examples is the H3.3 gliomas . This mu gliomas . Howeverll death . TherefoAside from ERVs, numerous studies have linked LINEs and SINEs to a viral mimicry response . In factThe mechanisms that dictate whether elevated dsRNAs induce sublethal or lethal IFN responses remain unclear. Survival of the subset of cancer cells with both high dsRNA levels and ADAR1 deficiency suggests that ADAR1-mediated editing is not the only mechanism cancer cells utilize to evade immunogenic cell death. As we strive to develop more robust and clinically relevant drugs, other RNA regulators and editors providing compensatory immunosuppression must also be explored. One recently found example is the RNA helicase DHX9 in breast cancer cells .Long read methylome data indicates that hERV CpG methylation is lower at baseline than other TEs and the remaining genome in normal tissues . Thus, iPost-transcriptional methods of viral mimicry induction are undergoing further study for recurrent cancers like glioblastoma. Despite its promise, the potential benefits of leveraging inducible hERV activity against solid tumors must be closely balanced with possible unintended consequences such as ERV onco-exaptation.Several drugs, such as 5-azacytidine and decitabine inhibiting DNA methylation, are FDA-approved for hematological tumors. Of the many similarities and parallels between DNA and RNA methylation, the most similar is m5A. Interestingly, studies show the clinical response to hypomethylating therapies do not correlate with DNA methylation status, and approximately 90% of 5-azacytidine is incorporated into RNA . Thus, tThough no FDA-approved drugs currently target epitranscriptomic regulators, there is preclinical evidence of the anti-tumor effects of RNA-modifying therapy and a growing cadre of interested biotech companies . Most smUtilizing glioblastoma stem cells (GSCs) and xenograft mice, MA2, the ethyl ester form of meclofenamic acid (MA), an FDA-approved non-steroidal anti-inflammatory drug, was identified as a selective inhibitor of FTO that increases m6A mRNA levels and suppresses GSC-initiated tumor progression , 106. Si


The Canadian drug supply has significantly increased in toxicity over the past few years, resulting in the worsening of the overdose crisis. A key initiative implemented during this crisis has been data monitoring and reporting of substance use-related harms (SRH). This literature review aims to: (1) identify strategies used for the meaningful engagement of people who use drugs (PWUD) in local, provincial, and national SRH data system planning, reporting, and action and (2) describe data monitoring and reporting strategies and common indicators of SRH within those systems.We searched three academic and five gray literature databases for relevant literature published between 2012 and 2022. Team members who identify as PWUD and a librarian at Public Health Ontario developed search strings collaboratively. Two reviewers screened all search results and applied the eligibility criteria. We used Microsoft Excel for data management.n = 10 and gray literature reports n = 12); most used qualitative methods and focused on the Canadian context (n = 20). There were few examples of PWUD engaged as authors of reports on SRH monitoring. Among information systems involving PWUD, we found two main strategies: (1) community-based strategies  and (2) public health-based data monitoring and communication strategies . Substance use-related mortality, hospitalizations, and emergency department visits were the indicators most commonly used in systems of SRH reporting that engaged PWUD.Twenty-two articles met our eligibility criteria (peer-reviewed This review demonstrates limited engagement of PWUD and silos of activity in existing SRH data monitoring and reporting strategies. Future work is needed to better engage PWUD in these processes in an equitable manner. Building SRH monitoring systems in partnership with PWUD may increase the potential impact of these systems to reduce harms in the community.The online version contains supplementary material available at 10.1186/s12954-023-00902-x. The Canadian drug supply has significantly increased in toxicity over the past few years, driven by fentanyl and other synthetic opioids . This inDuring this crisis, one of the key actions implemented across the USA, Canada, and other international jurisdictions has been the monitoring of data on substance use-related harms (SRH)  to inform response efforts and strategies –7. RobusThis rapid review aims to summarize the peer-reviewed and gray literature on practices used to engage PWUD in monitoring of SRH. Specifically, our objective was to identify strategies used to engage PWUD in local, provincial, and national SRH monitoring and reporting systems. As a secondary objective, we describe existing data monitoring and reporting strategies that include community engagement, as well as common indicators used within these systems. Understanding current practices and indicators will help improve the meaningful engagement of PWUD in local and provincial SRH data monitoring for action in Ontario, Canada. We conducted a rapid review to document practices used to engage PWUD in local, provincial, and national systems to monitor SRH. Rapid literature review methods are often used to gain an understanding of what evidence exists about a particular topic and can be useful in developing research questions or methodologies . CompareThe review consisted of both peer-reviewed and gray literature searches. A librarian at Public Health Ontario (PHO) provided support for developing search strings, which included concepts such as “people who use drugs,” “overdose,” “hospitalization,” and “surveillance”  English language texts; (2) peer-reviewed and gray literature research manuscripts or reports; (3) focused on people who use unregulated drugs; (4) described strategies to engage PWUD in data monitoring and reporting on SRH; and (5) were published between the years of 2012 and 2022 (current at the time of the search). The search included documents from the last ten years to ensure relevancy to current data monitoring and reporting processes. In Canada, OxyContin was delisted from provincial drug funding formularies as of March 2012. This coincides with the acceleration of fentanyl-related deaths from 25.8% of opioid-related deaths in Ontario in 2012 to nearly 90% by 2021 , 14. ArtTwo reviewers  applied the eligibility criteria to the titles and abstracts in pairs, as outlined above. The team collaboratively reviewed 20 articles (10 peer-reviewed and 10 gray literature) to ensure consistency and to develop a common understanding of the eligibility criteria among all screeners. Articles that met the above eligibility criteria based on titles and abstracts subsequently underwent full-text screening. The review of full-text articles followed the same process as titles and abstracts. A third reviewer was involved in discussion of all discrepancies among screeners. We used a screening template in a Microsoft Excel Spreadsheet as a structure for data management with all articles, which included information such as the article title, abstract, and year, the search engine used to find the source, and reasons for exclusion.We used a structured table in Microsoft Excel to manage data extraction. One team member (MP or DEOP) reviewed each article and extracted the following information: location of work, data collection and analysis methods reported, and engagement strategies described in the text. We also extracted information pertaining to data monitoring and reporting strategies  and any indicators used for specific outcomes that were identified within the literature. The co-principal investigator (PL) reviewed each of the extracted articles to ensure consistency. Once the screening and extraction steps were complete for each article, the full team met three times to analyze and interpret the findings. We developed a typology to apply to the engagement strategies described in the included manuscripts that distinguishes the roles of PWUD, with definitions described in Table n = 12). The majority of literature focused on the Canadian context (n = 20) with one originating from the USA (n = 1) and another from North America  (n = 1). All articles were published between the years of 2015 and 2022, with most being published between 2019 and 2022.Our search results consisted of 6727 records , and 22 articles met the eligibility criteria. See Fig. There was limited discussion of specific strategies used to engage PWUD within SRH data monitoring and reporting systems. Levels of engagement among PWUD varied across the included articles. One article did not involve PWUD directly in the development of the review but included literature on information and risk communication strategies among PWUD on drug adulteration and quality, relevant SRH monitoring and reporting systems . Table 2n = 11) in studies related to monitoring and reporting systems. Three articles engaged with PWUD as advisory members of the systems [There were four main ways in which PWUD were engaged in SRH data monitoring and reporting systems: as participants in research studies, as advisory members, as consultants, or as co-authors. People may have been engaged in more than one role. Most of the academic literature included in this report engaged PWUD as participants  and communication methods for sharing information among PWUD, including current indicators used in data monitoring systems. Among the gray literature reports, four reports described jurisdictional strategies and action plans aimed at reducing opioid or broader SRH . These included community recommendations on timely and effective communication of drug alerts and online dissemination of information.Eleven articles focused on community-based strategies for monitoring and reporting SRH , 29–37. n = 32) agreed that “word-of-mouth” strategies were most commonly used for gathering and providing information about drug quality [Peer-based social networks were also described as a drug monitoring and communication community-based strategy. Soukup-Baljak et al. (2015) discussed the methods of communicating drug quality to PWUD in British Columbia, Canada, and found that all participants  focused on local strategies and plans targeted at minimizing SRH  [Eight articles included in this review leveraged public health-related monitoring strategies for SRH –25, 33. esponse) –25. The Provide anonymity or confidentiality.Be easy to use.Be a place to find archived and factual information.Be able to reach a large number of people.Be a place to report drug-related concerns .Be a place to share personal stories.The York Region Opioid Action Plan, for example, indicates that a local warning system aimed to share information about overdoses and other SRH should be developed and shared among varying health and social service providers . This acThe recommendations from York Region also addressed the importance of incorporating indicators within monitoring of SRH that are informed by people with lived/living experiences . Consultants noted that indicators such as emergency overdose visits or hospitalizations neglect to consider the realities of opioid-related harms in communities  and ignore the fact that many PWUD avoid hospital settings due to experiences of stigma and harm.Timeliness is a key consideration for the effectiveness of communication such as drug alerts.Neutral and trigger free language should be included in communication.Most jurisdictions globally  have incorporated web or app based strategies for monitoring and communication of SRH.Poster communication is valuable but not as accessible as “word-of-mouth” communication which reaches groups not connected to health or social services. This indicates the need for varying forms of communication pertaining to SRH.Incorporating people with lived/living experience in the creation and dissemination of SRH communication is essential.Similar strategies were discussed within the Toronto Overdose Action Plan: Prevention and Response, which also consulted people with lived/living experience . RegardiThe Peel Opioid Strategy: A Local Response document  and a reAmong systems that engaged PWUD, six articles included in this review investigated SRH trends in by region, composition of substances, or characterized the circumstances surrounding overdose events. Literature published over the last two years (2020 and 2021) focused on how trends were influenced by the COVID-19 pandemic , 27. ThrIn addition to the above documented SRH indicators, the relevant literature also focused on reporting the quality and consistency of substances in particular jurisdictions. Scarfone et al. (2022), for example, investigated the chemical composition of high-potency opioids, such as fentanyl, being consumed in Toronto, finding 87% also contained stimulants, while 21% of samples included benzodiazepine-type drugs, and 1% included synthetic cannabinoids . At the This literature review found a total of 22 articles, ranging from peer-reviewed to gray literature reports that engaged PWUD in local, provincial/state, and national SRH data system reporting. We found little detail on the engagement strategies used, with several articles involving PWUD as research participants and few articles with involvement as authors.Our results demonstrate that the limited engagement of PWUD as leaders in the development and implementation of SRH reports represents a major limitation in the existing literature and current documented practices. As an interdisciplinary group grounded in the principal of “nothing about us without us,” we believe that without meaningful involvement of PWUD in leadership positions , existing SRH monitoring and reporting strategies will be unsuccessful in preventing morbidity and mortality, and risk furthering individual and system-level stigma and harm . In contThe Nameless, a community-based, peer-led harm reduction organization in St. Thomas, Ontario, that aims to provide support to help lessen harms associated with varying forms of poverty [Information on community-based/peer-led initiatives may not be publically available and thus not captured in our review, although we are aware of examples that address the issues outlined above. One example that community experts referred to was  poverty . They woOther initiatives such as the Real-Time Drug Alert and Response project , locatedThere remains a need to further reduce barriers to engagement for PWUD in adapting and developing future SRH data monitoring systems. Our community-based team members have identified that to build the capacity of existing community networks larger government run monitoring systems must: (1) increase funding to community-led groups to advance strategies currently being used for SRH data monitoring and communication; (2) remove barriers faced by PWUD such as stigmatized hiring practices ; (3) employ and provide training to PWUD as core stakeholders in provincial, territorial, and federal SRH data monitoring and communication agencies; (4) hold organizations accountable to existing best practice guidelines for engaging PWUD; and (5) commit to working across relevant stakeholders to use data for action to reduce harm associated with substance use. These suggestions reflect the application of Canadian best practice guidance on meaningful engagement of PWUD, addressing issues such as funding, criminalization, and employment, “to ensure equitable and just opportunities within program and policy domains that affect their lives” .Existing guidelines and research addresses strategies that can help facilitate the engagement of people with lived/living experience in the substance use sector . For exaPWUD face multifaceted forms of oppression by existing systems that drive current SRH monitoring and communication. In addition, people with living/lived experience leading work within local systems are under immense pressure and face high levels of trauma and grief from the continued loss of friends, family members, and peers , 51. To Further research is needed on how best to operationalize expert knowledge in a way that is equitable and truly meaningful in monitoring and reporting of SRH. Effort must also be placed on shifting existing ideologies surrounding SRH data to allow PWUD to inform, develop, disseminate, and take ownership of monitoring and dissemination systems. As an interdisciplinary group of experts, we note the following considerations for future work in this area based on existing evidence and practice: (1) increase networking and support among relevant SRH data monitoring stakeholders ; (2) create processes for collecting and disseminating SRH that can be used by all community and public health-based organizations; and (3) develop an interactive map which shows drug alerts in different jurisdictions that can be accessed provincially by coalitions of PWUD  , 53. ForEven though this literature review was carried out using rigorous methods, it has limitations. Primarily, a large portion of literature included in this review was based in Canada within the provinces of Ontario and British Columbia. Including a search of local public health unit websites in Ontario likely over-represented information in this context ; however, all other searching encompassed provincial/state, national and international levels. This focus in Ontario and British Columbia does not speak to current strategies which may be used across North America or in other parts of Canada, for the engagement of PWUD in data monitoring and reporting. Similarly, literature included here was limited to the English language and to publically available information . The search was completed at an earlier stage of a larger project and the capacity to update the search was limited. Further, public health resources at the national, province/state, and local level were largely consumed with response to the COVID-19 between 2020 and early 2023. While there have been resources for interventions to mitigate SRH during the pandemic, there has not been a focus on evolving data monitoring systems during this period.In addition, there are a variety of different strategies which were highlighted in this review but our search did not identify information on the impact or effectiveness of systems for SRH data monitoring. Finally, although out of scope of this review, there were no methodological assessments completed of included studies. Despite this, our review presents unique findings surrounding current engagement of PWUD in SRH data monitoring with leadership from team members with lived/living experience of substance use.Effectively engaging PWUD in SRH data monitoring and reporting is essential to improve effectiveness and mitigate ongoing harms associated with the current overdose crisis. There remains limited engagement of PWUD as authors or leaders in existing data monitoring and reporting practices. Currently, data monitoring and reporting strategies which take place in the community and within broader public health sectors are siloed. Considerations for how to effectively engage PWUD in these processes are needed and to improve capacity of existing community-run data monitoring and communication systems.Additional file 1. Appendix 1.Additional file 2. Appendix 2.


Aedes albopictus is an aggressive invasive mosquito species widely distributed around the world, and it is also a known vector of arboviruses. Virus metagenomics and RNA interference (RNAi) are important in studying the biology and antiviral defense of Ae. albopictus. However, the virome and potential transmission of plant viruses by Ae. albopictus remain understudied.Ae. albopictus were collected from Guangzhou, China, and small RNA sequencing was performed. Raw data were filtered, and virus-associated contigs were generated using VirusDetect. The small RNA profiles were analyzed, and maximum-likelihood phylogenetic trees were constructed.Mosquito samples of Ae. albopictus revealed the presence of five known viruses, including Wenzhou sobemo-like virus 4, mosquito nodavirus, Aedes flavivirus, Hubei chryso-like virus 1, and Tobacco rattle virus RNA1. Additionally, 21 new viruses that had not been previously reported were identified. The mapping of reads and contig assembly provided insights into the viral diversity and genomic characteristics of these viruses. Field survey confirmed the detection of the identified viruses in Ae. albopictus collected from Guangzhou.The small RNA sequencing of pooled Ae. albopictus in this study sheds light on the diversity and prevalence of viruses in mosquito populations. The presence of known and novel viruses highlights the need for continued surveillance and investigation into their potential impact on public health. The findings also emphasize the importance of understanding the virome and potential transmission of plant viruses by Ae. albopictus.The comprehensive analysis of the virus metagenomics of Ae. albopictus and its potential role as a vector for both known and novel viruses. Further research is needed to expand the sample size, explore additional viruses, and investigate the implications for public health.This study provides valuable insights into the virome of  Aedes albopictus, originating from the forests of Southeast Asia, was first recorded on La Réunion Island in 1913  are conserved, sequence-specific gene regulation mechanism in eukaryotes, including insects  was used to grind the samples with 200 μl of TRIzol reagent  on ice. The sample homogenate was then clarified by centrifugation at 20,000 ×g (4°C) for 30 minutes and filtered through a 0.45 μm membrane filter . The TruSeq Small RNA Sample Prep Kit  was used to prepare the sRNA library with 1 μg of total RNA as input, following the manufacturer’s instructions. Briefly, polyacrylamide gel electrophoresis (PAGE) purification of RNA bands, enriching RNA molecules in the size range of 16–50 nt the small RNAs, were preferentially ligated with 3’ and 5’ adapters, reverse transcribed using Superscript II reverse transcriptase , and PCR amplified. During the amplification process, a unique oligonucleotide barcode sequence was incorporated into each library for multiplexing purposes. The resulting small RNA libraries were then size-selected on 2% TBE-agarose gels and purified using MinElute Gel Extraction kits (Qiagen). The purified cDNA libraries were eluted in water and quality-checked on the 2100 Bioanalyzer. The small RNA libraries were sequenced using SE50 by Illumina Nextseq 500 according to the manufacturer’s instructions. Small RNA library sequencing was completed by Genedenovo .Ae. albopictus genome as reference sequence. Small RNA length distribution graphs and viral genomic position maps were produced using the viRome package (http://www.ark-genomics.org/bioinformatics/virome) in R. The sequences from the small RNA libraries of all nine pools were submitted to the NCBI Sequence Read Archive (BioProject - PRJNA527345). Maximum-likelihood phylogenetic trees were constructed using the Mega 7 program , followed by siRNA (21-22 nt) and miRNA (24 nt). In this study, piRNA was the most dominant in all nine pools Figure 1Aedes flavivirus, Hubei chryso-like virus 1, and tobacco rattle virus RNA1.Mapping reads to the virus databases of NCBI indicated a mapping rate of 0.25-0.93% in the different pools Table 3.Wenzhou Sobemo-like virus 3 was identified in eight pools. Most Wenzhou sobemo-like virus 4 mapped sRNA were 21 nt in length , while others can enter the insect gut and circulate or replicate within the insect vector body  can be found below: X-GC conceived and contributed to the study concept and design. YX, JX, TL and PL collected the samples. YX and JX completed all analyses and finished the manuscript. X-GC supervised the study. All authors contributed to the article and approved the submitted version.


Serum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype  and HBeAg status.Longitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients  was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow.All chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time.This study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response. In addiWhile the clinical role of serum HBV RNA as a biomarker for monitoring CHB is highly studied, there remains a lack of understanding of the composition of serum-based RNA . ExploraThis study aimed to make preliminary observations of the serum HBV transcriptome of CHB patients having various clinical profiles and HBV genotype (gt) using Oxford Nanopore Technology (ONT) sequencing, and so provide the first detailed sequence description of serum-based HBV encapsidated transcripts.2.2.1.Specimens were collected from 13 HBsAg positive, CHB patients ; all patients were under the care of a hepatologist, except one individual (19/0039) who was an asymptomatic HBsAg positive carrier. 2.2.Total nucleic acid was isolated from 600 μL of patient serum using the EasyMAG extraction system , with no specific enrichment for virus-like or nonenveloped particles. Twenty microgram of yeast tRNA  was added to the lysis buffer and nucleic acid elution was in 60 μL of elution buffer. Detection and quantification of RNA after serum extraction were done using the High Sensitivity RNA ScreenTape system on the 2200 TapeStation  using the provided protocols with the 2,200 TapeStation Software (v A.02.02 SR1).E. coli Poly(A) Polymerase as per the manufacturer’s protocol . Both reactions were combined and purified using Agencourt RNA Clean XP beads . Elution and concentration of the combined reactions was in 11 μL of nuclease-free water.Total extracted RNA from serum or plasma was assumed to be the fraction containing encapsidated, polyadenylated and non-polyadenylated, HBV RNA species. In order to capture possible non-polyadenylated 3′ truncated variant transcripts, a total RNA extract of each sample was also subjected to polyadenylation. Polyadenylation reactions were prepared in order to have a total volume of 40 μL  in order to compensate for the additional manipulation and purification required for polyadenylation. Briefly, each 20 μL reaction included treatment of 8 μL of total RNA extracted from patient serum with an 2.3.The protocol provided by ONT  for the Direct RNA sequencing kit  was used. Libraries were loaded on R9.4.1 MinION Flow Cells . Sequencing runs were 24 h.2.4.The protocol provided by ONT for the PCR-cDNA Barcoding kit  was used. The PCR step had 14 cycles of 3.5 min. Libraries were loaded on R9.4.1 MinION Flow Cells. Sequencing runs were 32 h.2.5.To estimate the basecall error of RNA sequenced by the ONT PCR-cDNA sequencing kit, a pgRNA transcript, transcribed from a T7 polymerase DNA vector  containing an HBV gtA 1.1x HBV genome insert (GenBank accession no. OQ54047) was polyadenylated as described previously. The polyadenylated pgRNA transcript was reverse transcribed and PCR amplified with SP6 or − 48 M13 rev primers to allow Sanger sequencing by primer walking . The polyadenylated transcripts were also sequenced using the Nanopore PCR-cDNA kit.The combined FASTQ file from PCR-cDNA sequencing was processed in Pychopper (v 2.5.0) with default parameters and in Filtlong (v 0.2.1) using --min-length 500, −-min-mean-q 80 (percent sequence identity) and --max-length 3,400. Bowtie2 (v 2.3.4.3) was used to align the nanopore filtered read file to each of the assembled Sanger sequence contigs. The BAM2 mapping statistics tabular file output was used to assess the mean nucleotide variant percentage across approximately 3.1 kb to calculate the error rate of long-read sequencing. The mean error rate calculated among each of the three reference contigs was deemed the PCR-cDNA sequencing error.2.6.Each of the 32 RNA preparations for PCR-cDNA sequencing  were spiked with spike-in RNA variant (SIRV) Set-4 RNA spike-in variant mixtures  and the University of Manitoba institutional ethics review board (protocol ID H2020:403).2.9.p-values below 0.05.Correlations between expected and measured values of SIRV control attributes were evaluated using Pearson’s correlation coefficient within SIRVsuite . Kruskal2.10.Raw data FASTQ files were submitted to the NCBI Sequence Read Archive under BioProject ID PRJNA853798. HBV reference and pgRNA sequences used for mapping and error determination were submitted to GenBank (accession nos. OQ054047-OQ054050).3.3.1.Initial sequencing attempts using direct RNA sequencing (dRNA-seq) were unsuccessful due to the low quantity of reads mapping to HBV. An HBV gtC serum pool was extracted and concentrated to compare dRNA-seq and PCR-cDNA sequencing. Resulting FASTQ files were mapped to a theoretical pgRNA reference based on GenBank accession number AF223954 in Minimap2. While the dRNA-seq run returned 11 HBV RNA reads within the output .bam file , the PCR-cDNA run returned 3,226 HBV reads . Thus, PCR-cDNA sequencing was considered more effective than dRNA-seq at obtaining higher read depth for HBV RNA isoform detection from low biomass specimens.Sixteen samples from 13 patients were subsequently processed using PCR-cDNA sequencing methods. Total RNA and a polyadenylated reaction product from each patient RNA extract  were sequenced. The raw combined FASTQ file data for all samples had a mean read length, mean read quality (Phred score), and length N50 of 0.657 kb, 10.48, and 0.722 kb, respectively, as determined by NanoPlot analysis, which is consistent with median transcript read lengths between 0.7 and 1.8 kb reported in the literature . The meaHepadnaviridae (HBV), human (SIRV spike-in control RNA), and non-specific  and non-classified. Using the Kraken 2 data from all total RNA reads , the median proportion of reads for 16 samples was 0.08% for HBV, 31% for human, 49.5% for non-specific, and 19.4% for non-classified. These results, corroborated by Bracken (The Kraken 2 standard database (created 2021-05-17) was utilized to determine reads corresponding to  Bracken  as mean Pearson’s R values for ERCC  control isoforms generated by SIRVsuite software ranged from 0.74 to 0.88 and the mean value for all sequenced samples was 0.82. ERCC control RNA allows a measure of isoform abundance complexity, limit of detection and an assessment of workflow efficiency . These Rp > 0.99 in Dunn’s multiple comparisons test) and samples within showed significantly similar performance, with the exception of some discrepancies observed between samples from the same run . All three pipelines detected full-length pgRNA transcripts within the total RNA extract , althougThe detection of putative transcripts within the total RNA extract was also quantified by each workflow to account for possible polymerase drop-off during nanopore library preparation or RNA degradation during extraction, although there is the possibility that putative transcripts were artefactual truncated transcripts. Putative pgRNA was defined by a length > 1.2 kb and the presence of the 3′ sequence signature. All methods produced clusters of reads (<1.2 kb) from the direct RNA extract having the expected pgRNA 5′ end, but with irregular or truncated 3′ ends. These may have been due to fragmentation or mis-priming of the non-specific poly(T) primer used for reverse transcription during library preparation , and thuA variety of pgRNA spliced variants were detected with all three methods originating from the total RNA extract and the polyadenylated products, although the Galaxy workflow, which incorporates Kraken 2, GraphMap, and length filtering tools within the online Galaxy infrastructure, was the only method providing complete spliced transcripts . SplicedThe main differences between the isoform detection methods were the identification of 3′ truncated variants and HBx transcripts. The Galaxy workflow method was the only pipeline to detect full-length HBx mRNA transcripts having variable 5′ sequence coordinates, ranging from nt 1,273 to 1,328, while FLAIR identified a transcript matching the HBx gene reference coordinates , most likely due to the very low proportion of HBV specific reads detected among total nanopore reads in total extracted RNA , nor was any specific trend in detected isoforms observed among HBeAg positive and HBeAg negative samples.Sequenced samples were arranged into groups based on HBeAg positive or negative status. Two samples were HBeAg negative, H21/3966 and H21/5698, both HBV gtA. HBeAg positive samples comprised gtB , gtC , and gtD , and gtF (291). Consistent detection of specific full-length transcripts was not observed in either group, depending on the isoform identification method . No sign3.5.Three patients, LU223, LU233, and LU279, were longitudinally sampled pre- and post-partum (~7 months between time points), as a preliminary investigation of serum HBV RNA isoform trends over time . Read co4.Serum HBV RNA has been shown to be a valuable surrogate biomarker of intra-hepatic cccDNA levels in monitoring of CHB infection. More specifically, it has been used to monitor response to treatment , to pred4.1.Pregenomic RNA was repeatedly detected among samples , either as a transcript of full-length (~3.33–3.34 kb) or a partial transcript termed “putative” as it contained the pgRNA 3′ sequence signature but was of variable length > 1.2 kb. pgRNA has been described to comprise the greatest proportion of serum HBV RNA . Both RAA striking observation from our HBV isoform identification data is the absence of cryptic trRNA. This transcript isoform, having a 3′ truncation due to the presence of a cryptic polyadenylation signal upstream of the canonical poly(A) site, was firstly observed in tumor and non-tumor tissue from patients with HCC . It is tFollowing synthetic polyadenylation of serum HBV total extracted RNA, pgRNA 3′ truncated variant transcripts were identified in 14 of 16 clinical samples. These transcripts contain the 5′ sequence signature of pgRNA transcripts but are not full length, with variable length greater than 1.2 kb. The 3′ truncated variant transcript proportion of HBV reads within polyadenylated RNA products varied depending on the isoform detection method, but comprised the majority of transcripts identified in several specimens. Indeed, a 3′ truncated variant was the sole transcript detected in one specimen , as identified by both FLAIR and Galaxy workflows. It has been reported that many HBV RNA species secreted in serum are non-polyadenylated , with pgAn interesting observation was the myriad of pgRNA spliced variants identified with all three isoform detection methods, including Sp1, Sp5-6, Sp9-10, Sp12, Sp20-22, pSp7, and pSp11 . AdditioHBx full-length transcripts having a reported length between 0.7 and 0.9 kb  with 5′ The use of a SIRV control allowed for measurement of accuracy, precision, and inter-experiment comparability , the lat4.2.Previous studies have looked at differences in serum HBV RNA by HBV genotype and have found that gtB, C, D, and F expressed higher levels of pgRNA, whereas gtA expressed the lowest . SimilarPatient sample RNA transcripts were analyzed based on HBeAg serostatus. Very few studies have evaluated serum HBV RNA levels and profiles as a function of HBeAg presence or ALT elevation ; howeverLongitudinal samples were collected from three NA-treated individuals pre- and post-partum to allow preliminary observations of serum HBV RNA over time. Very few studies have looked at the dynamics of HBV RNA composition and quantity in clinical samples, but it is known that spliced variant diversity increases over time . No sign4.3.HBV full genome sequencing studies, or those exploring the HBV transcriptome, have largely employed PCR- or amplicon-based deep sequencing methods . To datede novo isoform identification method was important. However, RATTLE often mis-identified the expected pgRNA 5′ sequence signature due to HBV having two direct repeats, DR1  and DR2 , employed during HBV replication. The pgRNA “true” 5′ end is associated with DR1, and thus, recognition of the upstream DR2 site created RATTLE-identified transcripts that were longer than expected, having a 5′ end at approximately nt 1,590. RATTLE tends to merge very similar reads into a single transcript and is thus more conservative in terms of the number of predicted known or novel transcripts identified.There were distinct differences in the isoforms detected and their proportionality depending on the analysis pipeline used. FLAIR and RATTLE are mostly used for transcript identification in eukaryotic cells and RNA spike-in material , and theWhile RATTLE’s precision was found to be lower than reference-based methods such as FLAIR and StringTie , we obseThe fact that FLAIR requires an annotated reference file posed a challenge, as no annotated HBV genomes with all expected transcript isoforms exist in present databases. The choice of reference highly influences the efficacy of transcript identification by FLAIR, which was the most effective method for the identification of known and novel spliced variants. Validation of splice junction sites, such as comparison to short read Illumina data  was not While evaluating the long-read sequencing data, the presence of co-morbidities and the clinical status of the HBV-infected patient is important to consider. The primary purpose of serum HBV RNA quantification is for clinical applications; however, the composition and proportion of each serum RNA species may differ depending on the clinical profile . The imm4.4.These results present for the first time, initial observations of the serum-based HBV transcriptome by long-read sequencing technology among longitudinal and cross-sectional specimens of patients having various hepatitis B clinical profiles and genotypes. Pregenomic RNA was observed to be the major transcript detected among patients infected with various HBV genotypes (A to F), although the proportions of serum-based HBV transcripts detected by cDNA-PCR nanopore sequencing differed dependent on the sequence analysis workflow. The results obtained from long-read sequencing of the HBV transcriptome illustrated the strengths and challenges of using nanopore long-read technology and the utility of several bioinformatics methods for serum HBV RNA isoform sequencing and quantification. The study provided novel insights into the profiles and proportions of HBV RNA isoforms in chronic infection to support further understanding of this biomarker for management of CHB patients.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI - PRJNA85379.The studies involving human participants were reviewed and approved by the Health Canada and Public Health Agency of Canada REB (protocol ID REB 2019-036P) and the University of Manitoba institutional ethics review board (protocol ID H2020:403). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CO and AV: conceptualization, formal analysis, funding acquisition, methodology, and writing. GS, AV, NP, and EM: data acquisition. CO: project administration and supervision. AV, GS, and CO: result analysis and interpretation. CO, CC, EM, and EE: laboratory and software resources. All authors reviewed, revised, and approved the manuscript.AV was supported by a 2020 Canadian Liver Foundation Graduate Studentship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications. Small Cas enzymes are required for therapeutic use. Here the authors report an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturisation and use this to generate five compact variants of Cas13 based on a combination of IDC strategy and AlphaFold2. However, the CRISPR‒Cas editing systems for the therapeutic applications, along with the promoter, expression regulatory elements and other functional proteins, remain challenging due to the in vivo delivery constraints of adeno-associated virus (AAVs)13. The small Cas enzymes have attracted considerable attention as they transcend protein size restrictions for use in broad applications, such as CasMINI14, Cas12f15, OMEGA16, CasX18, CasY18, CasΦ19, mini-dSpCas9:VPR, and mini-SaCas9:VPR20.The CRISPR‒Cas system has been developed to be a revolutionary gene-editing technology that shows precise targeting, high efficiency, and programmable DNA- or RNA-targeting properties22. However, these compact Cas proteins do not completely recapitulate the advantages of previously optimized Cas proteins for example. Cas13bts show less efficiency than RfxCas13d for RNA transcript knockdown. In another strategy of miniaturization, the functionalities of Cas enzymes are modified. However, in this strategy, the Cas DNA-/RNA-cleavage activity is commonly eliminated, whereas its DNA-/RNA-binding activity is preserved. For example, base editors, such as HNHx-ABE and xABE, which remove a functional domain, are typically constructed by maintaining deaminase and DNA-/RNA-binding activity23. Additionally, MISER presents the overall absence of SpCas9 by generating a library of random deletion mutants, enabling Δ3CE and Δ4CE constructs to retain only approximately 72% and 63% of the primary dead SpCas9 protein sequence, respectively, with a commensurate decline in DNA-binding and cleavage activity24. Furthermore, although a previous study has suggested that constructing surface-localized deletion variants of poorly conserved Cas13d orthologs allow protein size reduction with minimal loss of efficiency, this process is largely restricted by the available fragments being unable to meet the miniaturization requirements of a Cas protein for future applications25. Because of the aforementioned challenges, it is necessary to develop a miniaturization strategy for in vivo gene therapy.Previous studies have shown several successful strategies used to miniaturize Cas proteins. An effective method is initiated by searching for natural compact Cas enzymes in metagenomic datasets, such as the 775 amino acids (AAs) Cas13X.1 and 790 AAs Cas13bt129 and is classified into six subtypes, Cas13a-d, Cas13X and Cas13Y. Functionally, all Cas13 effectors carry a pre-crRNA processing catalytic center and a target RNA cleavage catalytic center formed by two conserved R-X4-H motifs30. In addition, Cas13a and Cas13d both comprise a crRNA recognition (REC) lobe and a nuclease (NUC) lobe, which exhibit similar conformational changes from the binary surveillance complex to the ternary complex; for example, the binding channel is enlarged to accommodate the crRNA-target RNA duplex and the distance of the R-X4-H motif between the HEPN1 domain and HEPN2 domain is reduced to activate the HEPN nuclease active site33. Differently, the open conformation of HEPN1 and Helical-2 is the major conformational change of Cas13b from binary to ternary35. Furthermore, the results of thermal denaturation assays indicated that HEPN1 and Helical-2 assume an open conformation to allow target RNA to enter the Cas13b central cavity, initiating target recognition starting from the 3′ side of the spacer sequence in PbuCas13b35. This open conformation was demonstrated in the Cas13bt3-crRNA-targetRNA ternary complex36.The CRISPR‒Cas13 system has been described as a crRNA-mediated CRISPR nuclease that exclusively targets RNAThus, we propose an IDC strategy for protein miniaturization with the goal of maximizing protein miniaturization while preserving protein function based on protein structure in this study.29. Conformational degrees of freedom describe fluctuations around a native conformation and switching to functional states37. In the Cas13-crRNA-target RNA ternary complex, sites with an effective degree of freedom are concentrated in crRNA-target RNA contact sites, pre-crRNA processing sites, and allosteric target RNA cleavage sites19. Therefore, we chose the secondary structural element with the highest effective degrees of freedom as the research object for observing distribution in the relative domain.Interaction refers to the interaction between functional sites of Cas enzymes Fig. . The Cas38. Upon crRNA binding and target RNA binding, domains are rearranged during the transition, resulting in a particularly noticeable change in the overall conformation of a domain; notably, the intradomain reorganization of secondary structural units is changed to a lesser extent29. The conformational variations of a domain contribute to the conformational variation in an entire protein, partly because of the long-range correlations in the native dynamics of proteins and local perturbations at any residue that can be sensed by other spatially distal residues in a protein molecule39. In addition, the portions of domain units are not essential to domain function; for example, HEPN domains can present morphologies that are tailored to protein function by integrating them into different units. The HEPN domain of Cas13a is larger than the normal HEPN domain because, in Cas13a, HEPN comprises numerous extra helices and a tiny hairpin insertion between α2 and α340. In addition, secondary structural units with low correlations and less dramatic functional conformational change for suitability as research objects are deleted during the intradomain reorganization of the Cas13 enzyme.Dynamics refers to the conformational change within a domain41. Although sequence homology is low, the structures of Cas13d protein family members display extraordinarily high similarity. For conserved structural units, analyzed deleted fragments are directly generalizable to the entire Cas13d protein family; for unique structural units of a particular Cas13d homolog, interaction and dynamics analyses are performed to evaluate whether they fulfilled the deletion criteria. Furthermore, the principle of fragment deletion is irrelevant to the conservation of protein sequences; even structures with highly conserved sequences can be considered for fragment deletion.Conservation refers to the conserved structural units of the Cas13d protein family, as well as the structure-specific units of a protein in the family. Attributing to the detailed prediction of the structure by AlphaFold2, conserved structural units in the Cas13d protein family and unique structural units of each Cas13d homolog are determinedPcsk9 transcript expression. In summary, by incorporating structural resolution and artificial intelligence, this strategy can be generalized to the entire Cas13 protein family.In this study, considering protein structure, we propose an IDC strategy for protein miniaturization based on the nucleoprotein interaction, dynamic conformation reorganization, and orthologs conservation. We apply the IDC strategy to generate five miniaturized variants of the Cas13 protein and verify that these ultrasmall variants of Cas13 all mediated effective mammalian transcript knockdown comparable to that of WT proteins. Furthermore, we engineer a compact RNA base editor composed of engineered ADAR and mini-RfxCas13d that exhibits robust editing efficiency relative to that of Vx and demonstrate packaging of mini-RfxCas13d within a single adeno-associated virus. Moreover, it efficiently knocks down To verify the optimization of the Interaction, Dynamics and Conservation (IDC) strategy, we designed eight deleted fragments in the closely related RfxCas13d ortholog utilizing the IDC strategy Fig. . The N-tThe HEPN1 catalytic domain of EsCas13d was the structural scaffold between the two lobes of Cas13d and the main domain constituting the crRNA channel. The region forming the central channel in HEPN1 had extensive interactions with mature crRNA and complementary target protospacers Movie . In contThe Helical-1 domain of EsCas13d underwent the most striking polypeptide rearrangement, moving outward to accommodate target RNA binding inside an enlarged cleft, though intra-domain reorganization in Helical1 was relatively little changed Movie . The intPortions of Helical-2 of EsCas13d showed subtle structural changes, particularly those close to the 3’ end of the spacer Movie , correspThe comparison of structural conservation of the Cas13d orthologs showed that α-helix11 of RfxCas13d was longer than that of EsCas13d in the Helical-1 domain Figs. , and S1828. The qRT-PCR results showed that all variants exhibited high knockout efficiency, with the efficiency of variants Δ4, Δ7, and Δ8 was not significantly different from that of RfxCas13d , thus establishing dmini-RfxCas13d-ADAR Fig. . Huang fAR Figs.  and S13.AR Figs. . As expesis Fig. . Overall48. To determine whether the efficiency of mini-RfxCas13d-mediated knockdown in vivo, a regulatory metabolism gene, the murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene, was chosen for our investigation. Specifically, we evaluated whether expression of Pcsk9 was suppressed by determining whether serum cholesterol levels were restored49. A previous study had demonstrated that the RfxCas13d system efficiently targeted RNA for knockdown in hepatocytes in vivo. Therefore, we first designed three crRNAs to target the coding sequence in Pcsk9 mRNA, as they had downregulated the level of Pcsk9 mRNA in a previous study (Table Pcsk9 crRNA were transfected into mouse neuroblastoma (N2a) cells that overexpressed a Pcsk9 mRNA fragment. As expected, all Pcsk9 crRNA candidates convincingly knocked down the Pcsk9 mRNA  driven by the CMV promoter to knock down Pcsk9 specifically in the liver. The construct was packaged into AAV8 and injected into the tail vein of 8-week-old mice , which is a combination of RfxCas13d and a deaminase domain inserted into the middle, is precise yet highly efficient, outperforming various previous versions with both mRNA and nuclear RNA targets36. Mini-Vx, a combination of mini-RfxCas13d and a deaminase domain inserted in the middle, was not only small enough to fit within an AAV vector but was also highly efficient.The IDC strategy offers many advantages compared with other methods of miniaturization. A multidimensional analysis of proteins preserved the RNA-binding and cleavage activity of the mini-Cas13 enzymes with little efficiency loss compared to that of the wild type Cas13 enzyme. The use of functional regions, in contrast to those described in the literature, enabled them to be directly studied as research objects, in contrast to studies based on changes in functionalities, such as the analysis of HNHx-ABE and xABE419. Therefore, this region was deduced to have exerted no effect on protein function and was therefore considered a deleted fragment.The IDC strategy is not based on the intersection of three IDC factors; in contrast, it is used to evaluate whether protein function is affected, which is critical to the analysis. The HEPN2 domain of RfxCas13d, for example, accommodates a larger insertion than RspCas13d and EsCas13d, which are located adjacent to the crRNA 5’ terminal DR region. The DR region of the crRNA 5’ terminus was exposed in the solvent, as determined during the structural analysis of EsCas13d, and exhibited no interaction with the protein29. Additionally, mini-RfxCas13d showed less collateral cleavage activity relative to RfxCas13d  mice were obtained from the Laboratory Animal Center of Jilin University . All animal studies were conducted under the guidance of the Animal Welfare and Research Ethics Committee at Jilin University (IACUC number: SY202204102).IDC strategy workflow of Cas13 is shown in Fig. 50. All animal studies were performed in accordance with the guide of the Animal Welfare and Research Ethics Committee at Jilin University.Institute for Cancer Research (ICR) mice were obtained from the Laboratory Animal Center of Jilin University 51. We compared the top five ranked outputs and selected Rank 1 to prepare the figures. Structural figures were generated with UCSF ChimeraX52.The whole structure of the Cas13d and Cas13b proteins was predicted by AlphaFold2 with Google Colab and default settings23. EsCas13d, RspCas13d, and PspCas13b sequences were amplified by PCR and cloned into a pcDNA3.1 backbone. Cas13X (#171379) and REPAIRx were gifts from Professor Hui Yang and Xingxu Huang, respectively38. Mini-RfxCas13d, mini-EsCas13d, mini-RspCas13d, mini-PspCas13b, mini-PbuCas13b, PbuCas13b and mini-Vx were synthesized and cloned into pcDNA3.1(+) . The Δ1, Δ2, and Δ3 RfxCas13d strains were constructed by homologous recombination. Δ4, Δ5, Δ6, Δ7, and Δ8 were synthesized and cloned into pcDNA3.1(+) by GenScript Biotech (China). The sequences of the proteins are listed in Table Cas13bt, EsCas13d, RspCas13d and PspCas13b were obtained from Addgene 2 in 6-well poly-D-lysine-coated plates (Corning). The cells were cotransfected with Cas13 (1500 ng) and crRNA plasmids (1500 ng) using Hieff TransTM Liposomal Transfection Reagent  according to the manufacturer’s instructions, and the cells were harvested 48 h later using TRIzol Universal Reagent . All Cas13 crRNAs used in this work are listed in Table HEK293T (ATCC) and N2A cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (HyClone) and incubated at 37 °C in 5% CO53. First-strand cDNA was synthesized using a FastKing RT kit (with gDNase) . Quantitative PCR (qPCR) was performed using QuantStudio3 (Thermo Fisher Scientific) with sample cDNA and SuperReal PreMix Plus (SYBR Green) . The primers are shown in Table −ΔΔCT method was used to analyze gene expression, and GAPDH was the reference gene. All experiments were repeated three times for each gene.RNA was extracted from successfully transfected cells following the description in a previous study28. Cas13d proteins were cloned into pET-28a with both N-terminal and C-terminal 6×His. The plasmids were transformed into Rosetta2(DE3) cells (Tolo Biotech), induced with 200 μM IPTG at OD600 0.5, and grown for 16 h at 16 °C. Cells were harvested by PBS supplemented with 1× protease inhibitor solution (Solarbio) and then sonicated and clarified via centrifugation , filtered with 0.45 μM PVDF filter and incubated with 1 mL of BeyoGold™ His-tag Purification Resin (Beyotime Biotechnology) overnight (at 4 °C). The column was balanced 2-3 times with 1 column volume of lysis buffer  before loading. The sample was applied to an affinity chromatography column, washed 5 times with 1 column volume of washing buffer  and 5 times with 1 column volume of Elution Buffer . Purified, eluted proteins were stored at 4 mg/mL in Protein Storage Buffer .The expression and purification of proteins were performed as described previouslyOligonucleotide sequences of crRNA, pre-crRNA, and ssRNA were synthesized (Sangon Biotech), annealed, PCR amplified and then subsequently purified respectively. The purified templates were in vitro transcribed with the Hiscribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and frozen at −80 °C.28. Briefly, Purified RfxCas13d, mini-RfxCas13d, PspCas13b, mini-PspCas13b proteins, and crRNA or pre-crRNA were mixed at a 1:2 molar ratio in RNA Cleavage Buffer . Before adding the target ssRNA at a 1:2 molar ratio in relation to the Cas13d proteins, the reaction was ready on ice and incubated at 37 °C for 15 min. After 45 min of 37 °C incubation, the reaction was stopped at 37 °C for 15 min with 1 L of an enzyme inhibitor solution  .After the reaction was denatured at 65 °C for 10 min with 2X RNA loading buffer , the sample was separated on a 15% TBE-Urea gel.The biochemistry cleavage reactions in vitro were performed as described previouslyhttp://baseeditr.com/)54. Deep sequencing was performed by using Hi-TOM (http://www.hi-tom.net/hi-tom/index-CH.php)55. All Cas13 crRNAs used in this work are listed in Tables Transfected cells were sorted for RNA extraction to assess the A-to-I base-editing efficiency of mini-Vx and Vx. A FastKing RT kit (with gDNase)  was used to reverse transcribe complementary cDNAs from RNAs, and crRNA target sites were amplified from cDNAs using 2*Taq PCR Master Mix II  for Sanger or deep sequencing. The Sanger sequencing results were analyzed with EditR to measure the effectiveness of the A-to-I conversion at each target site (N: 0.1). Using the program Hisat2 (v2.0.1), data were aligned to the reference genome. The DESeq2 Bioconductor package was utilized for the analysis of differential expression. Data-driven prior distributions are incorporated into the estimations of dispersion and logarithmic fold changes, and the Padj of the genes was adjusted to 0.05 to identify those that were differentially expressed.Forty-eight hours after transfection, total RNA was extracted from HEK293 cells. Stranded mRNA libraries were constructed by Genewiz and Novogene, and loaded on an Illumina HiSeq/Illumina Novaseq/MGI2000 instrument for sequencing using a 2 × 150 paired-end (PE) configuration according to manufacturer’s instructions. Fastq passes filter data were converted to high quality, clean data using Cutadapt .PackGene Biotech (China) created the AAV-mini-RfxCas13d-44. Production of AAV8-RfxCas13d vectors was performed by PackGene Biotech (China). The AAV-mini-RfxCas13d-Pcsk9-crRNA2 titer was 2 × 1012 genome copies (GCs)/mL. Before injection, all AAV doses were adjusted to a 100-μL volume with sterile PBS. For AAV8 vector injections, 8-week-old male ICR mice were given 2 × 1011 GCs per mouse intravenously via lateral tail vein injection. Twenty-one days after the injection, the mice were fasted for 12 h before liver and blood samples were collected. Housing conditions:12 h light/12 h dark, 23 ± 3 °C temperature, 50–70% humidity.An AAV injection assay was performed as previously describedg for 20 min. The serum was then poured into new tubes for further analysis. The total cholesterol, albumin (ALB), and alanine aminotransferase (ALT) levels in serum were measured with a Catalyst One chemical analyzer  following the manufacturer’s protocol44.Blood was collected from the eyeballs of mice. After standing at room temperature for 1 h, the whole blood was centrifuged at 2000 × Pcsk9 mRNA knockout levels confirmed .TRIzol  was used to extract RNA from liver tissue, and a FastKing RT kit (with gDNase)  was used to reverse transcribe the RNA. Thermo Fisher Scientific QuantStudio3 was used to perform quantitative PCR (qPCR) on liver tissue cDNA using SuperReal PreMix Plus (TIANGEN). AAV plasmid expression was measured, and med Fig. . Liver t56. Briefly, whole-cell extracts were prepared with RIPA buffer and phenylmethanesulfonyl fluoride  on ice for 30 min. The samples were vortexed briefly (10 min), and then subjected to western blot analyses with the anti-HA antibody , anti-GAPDH Polyclonal antibody . The images were captured on the Tanon 5200 Imaging System (Tanon).Western blotting was performed as previously describedp < 0.05). *p < 0.05, **p < 0.01, and ***p < 0.001.All data are expressed as the mean ± s.d. of at least three replicates in all experiments. The editing efficiencies of different Cas enzymes in HEK293T cells were compared with GraphPad Prism software 8.0.1. Statistical significance is indicated by a probability value of 0.05 (Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie1Supplementary Movie2Supplementary Movie3Supplementary Movie4Supplementary Movie5Peer Review FileSource Data File


Here, we report the genome sequences of 10 Carnation mottle virus variants. Six variants originated from a single proprietary carnation cultivar, and four were derived from four different proprietary cultivars. All variants showed nucleotide differences, but the last four did not show any variation at the amino acid level. Carnation mottle virus  is a single-stranded positive-sense RNA virus with four open reading frames (ORFs) (Dianthus caryophyllus (carnation), can result in mild mottling in young leaves but typically only faint chlorosis in mature leaves. Besides the CarMV genome reference sequence NC_001265.2, just 11 complete variant genome sequences are present in Genbank (s (ORFs) . Infecti Genbank .To gain deeper understanding of the CarMV genetic variation, we conducted small-RNA sequencing (sRNA-seq) on 10 asymptomatic carnation plants from various undisclosed commercial cultivars sourced from an anonymous plant-breeding company in the Netherlands. In carnation experiment 1 (CE1), six out of eight plants belonging to the same confidential cultivar were found positive for CarMV enzyme-linked immunosorbent assay (ELISA). In carnation experiment 2 (CE2), four distinct confidential cultivars that tested positive for CarMV ELISA were used. All samples were collected in February 2020 from a greenhouse complex situated in the southern region of the North-Holland province in the Netherlands. Genomes sequences of possible CarMV variants were determined by examining the siRNA response of the plants .https://shop.wur.nl/primediagnostics/carnation-mottle-virus-carmv.html) following the protocol provided by the manufacturer. Small-RNA was isolated by grinding a flash-frozen ±1 cm2 leaf fragment to fine powder using mortar and pestle, dissolving the powder in QIAzol Lysis Reagent (Qiagen) and enriching small-RNA using the miRNeasy Mini Kit (Qiagen). Barcoded small-RNAseq libraries were generated using Ion Total RNA-Seq Kit v2 and Ion Xpress RNA-Seq Bar Coding Kit (Thermo Fisher Scientific). Sequencing (150 bases) with an Ion Proton System using Ion PI-v3 chips and a Ion Chef System using Ion PI-Hi-Q-Chef Kit (Thermo Fisher Scientific) CE1 and CE2 yielded on average 5 (CE1) and 31 (CE2) million reads per sample.The ELISA assay was performed using the Prime Diagnostics  CarMV Kit , mutations were observed, ranging from 108 (2.7 %) to 141 (3.5%) nucleotides  to 23 (2.0%) amino acids. Although there are nucleotide differences between the four CE2 CarMV variants, they are identical at the protein level, whereas the six CE1 CarMV variants are different at the genome and protein level.