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We report complete coding sequences of four divergent Phasi Charoen-like viruses from mosquito RNA-seq data sets. Each genome includes L, M, and S segments with minor base ambiguities. These sequences expand known phenuivirus diversity and may aid future research on mosquito vector competence and dengue virus interactions.
Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.
Guide RNAs (gRNAs) are short mitochondrially encoded RNAs that contain the information for editing of messenger RNAs in Trypanosoma brucei. Although a great deal of work has focused on the utilization of gRNAs in editing, little is known about the turnover of gRNAs. In this report, we utilized in organello pulse chase and in vitro RNA decay experiments to directly examine gRNA turnover. We found that gRNAs are degraded by a biphasic mechanism. In the first step of decay, 3' gRNA sequences encompassing primarily the post-transcriptionally added oligo(U) tail are rapidly removed. This is followed by a second step, which entails a comparatively slower degradation of the encoded gRNA body. Decay of the 3' end of the gRNA is sequence specific, as it does not occur on oligoadenylated gRNAs. In contrast, the nucleotide composition of the 3' extension does not affect the rate of degradation during the second, slower, decay step. Finally, competition assays suggest that complete gRNA decay is mediated by two distinct enzymes, one of which simultaneously recognizes elements of the oligo(U) tail and the encoded portion of the gRNA. Overall, these results provide the first evidence for a gRNA-specific decay pathway.
CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.
MicroRNA (miRNA)-guided messenger RNA (mRNA) translational repression is believed to be mediated by effector miRNA-containing ribonucleoprotein (miRNP) complexes harboring fragile X mental retardation protein (FMRP). Recent studies documented the nucleic acid chaperone properties of FMRP and characterized its role and importance in RNA silencing in mammalian cells. We propose a model in which FMRP could facilitate miRNA assembly on target mRNAs in a process involving recognition of G quartet structures. Functioning within a duplex miRNP, FMRP may also mediate mRNA targeting through a strand exchange mechanism, in which the miRNA* of the duplex is swapped for the mRNA. Furthermore, FMRP may contribute to the relief of miRNA-guided mRNA repression through a reverse strand exchange reaction, possibly initiated by a specific cellular signal, that would liberate the mRNA for translation. Suboptimal utilization of miRNAs may thus account for some of themolecular defects in patients with the fragile X syndrome.
The snoRNA-LBME-db is a dedicated database containing human C/D box and H/ACA box small nucleolar RNAs (snoRNAs), and small Cajal body-specific RNAs (scaRNAs). C/D box and H/ACA box snoRNAs are part of ribonucleoparticles that guide 2'-O-ribose methylation and pseudouridilation, respectively, of selected residues of 28S, 18S or 5.8S rRNAs or of the spliceosomal U6 RNA. Similarly, scaRNAs guide modifications of the spliceosomal RNAs transcribed by RNA polymerase II (U1, U2, U4, U5 and U12) and are often composed of both C/D box and H/ACA box domains. However, some snoRNAs do not function as modification guide RNAs, but rather as RNA chaperones during the maturation of pre-rRNA. The database was built by a compilation of the literature, and comprises human sno/scaRNAs that were experimentally verified, as well as the human orthologs of snoRNAs that were cloned in other vertebrate species, and some snoRNAs that are predicted by bioinformatics search in loci submitted to genomic imprinting, but have not all been experimentally verified. For each entry, the database identifies the modified nucleotide(s) in the target RNA(s), indicates the corresponding predicted base pairing, gives a few pertinent references and provides a link to the position of the sno/scaRNA on the UCSC Genome Browser. The 'Find guide RNA' function allows one to find the sno/scaRNAs predicted to guide the modification of a particular nucleotide in the rRNA and spliceosomal RNA sequences. The 'Browse' function allows one to download the sequences of selected sno/scaRNAs in the FASTA format. The database is available online at http://www-snorna.biotoul.fr/. It can also be accessed from the human UCSC Genome Browser via the sno/miRNA track.
BACKGROUND: Bulky disease represents a clinically aggressive subset of diffuse large B-cell lymphoma (DLBCL) associated with adverse clinical outcomes. The aim of this study was to investigate the influence of oncogenic mutations and tumor microenvironment alterations on bulky disease in DLBCL. METHODS: We analyzed a cohort of 939 patients with newly diagnosed DLBCL. Using DNA (n = 934) and RNA (n = 524) sequencing, we compared oncogenic mutations and tumor microenvironment (TME) alterations based on tumor diameter, with cutoff values at 5.0 cm and 10.0 cm. Further stratification by mutations in key genes (CD58, STAT6, EBF1) correlated with tumor diameter revealed distinct transcriptomic and immunologic profiles. Subsequent single-cell RNA sequencing, guided by these mutational signatures, resolved the cellular heterogeneity within the TME. RESULTS: Integrative analysis revealed that tumor diameter correlated with increased incidence of mutations in CD58, STAT6, and EBF1; adverse genetic subtypes such as EZB-like MYC+ and TP53Mut; activation of oncogenic pathways (JAK/STAT, BCR, PI3K, and MYC); and an immunosuppressive tumor microenvironment. Notably, immune checkpoint molecules varied across the bulky stages, with CTLA-4, TIGIT, ICOS, and CD28 expression inversely correlated with tumor diameter, while CD70 and 4-1BBL expression positively correlated. Single-cell RNA sequencing further revealed mutation-specific tumor microenvironment insights. CD58-mutated tumor exhibited a profoundly immune-deserted microenvironment dominated by malignant B cells with minimal immune infiltration, whereas STAT6-mutated tumor was associated with increased fibroblasts and CD4 + T cells, particularly regulatory T cells (Treg) and Th1-like cells; EBF1-mutated tumor was characterized by increased proportions of malignant B cells. CONCLUSIONS: Collectively, our findings highlight the biological complexity of bulky disease, identifying candidate molecular targets and providing a biological framework for future therapeutic hypothesis generation in this clinically aggressive subset of DLBCL.
AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class II major histocompatibility complex (MHC II) molecules on cells. This paper studied the effect of Ribonuclease P (RNase P) against CIITA, which was a major regulator of MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte. METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli. It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoRI/BglII or EcoR//SalIsite of vector psNAV (psNAV-M1-452-GS, psNAV-M1-3408-GS) respectively. The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176). These recombinant plasmids were screened out by sequence analysis. psNAV-M1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro. It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS. Stable transfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class II MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RT-PCR., for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction. RESULTS: When induced with recombinant human interferon-gamma (IFN-gamma), the expression of HLA-DR, -DP, -DQ on psNAV-M1-3408-GS(+) hepatocyte was reduced 83.27 %, 88.93 %, 58.82 % respectively, the mRNA contents of CIITA, HLA-DR, -DP, -DQ and Ii decreased significantly. While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS(+) hepatocyte. CONCLUSION: The Ribonuclease P against CIITA-M1-3408-GS could effectively induce antigen-specific tolerance through cleaving CIITA. These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune diseases.
MOTIVATION: CRISPR-Cas9 genome-editing efficiency is strongly influenced by the sequence composition and positional context of single-guide RNAs (sgRNAs). Although numerous deep learning-based models have been developed to predict Cas9 efficiency from sgRNA sequences, most operate as black boxes, offering limited insight into the sequence determinants underlying Cas9 activity. In addition, previous studies often overlook how the positional context of sequence motifs within sgRNAs influences their effects on Cas9 binding or cleavage. RESULTS: We introduce DeepCC9, an interpretable machine learning framework that combines explicit sequence feature extraction with a residual block-based deep architecture to improve interpretability and identify composition- and position-based motifs governing Cas9 genome-editing efficiency. We applied this method to multiple Cas9 variant datasets, achieving superior predictive performance compared with existing methods while enabling direct interpretation of sequence motifs and their positional effects. Our analysis uncovered 74 sequence motifs enriched or depleted at specific positions within sgRNAs and strongly associated with Cas9 efficiency, providing mechanistic insight into sequence features that influence guide performance. Together, these results establish DeepCC9 as a generalizable and interpretable framework for modeling sequence-function relationships and advancing the understanding of the sequence determinants underlying CRISPR-Cas9 genome editing. AVAILABILITY AND IMPLEMENTATION: The authors have implemented their algorithm in the Python programming language (version 3.X), which is accessible using (https://zenodo.org/records/20073890).
BACKGROUND: Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. RESULTS: In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation. CONCLUSIONS: The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage.
Argonaute proteins are key components of the RNA-induced silencing complex (RISC). They provide both architectural and catalytic functionalities associated with small interfering RNA (siRNA) guide strand recognition and subsequent guide strand-mediated cleavage of complementary mRNAs. We report on the 3.0 A crystal structures of 22-mer and 26-mer siRNAs bound to Aquifex aeolicus Argonaute (Aa-Ago), where one 2 nt 3' overhang of the siRNA inserts into a cavity positioned on the outer surface of the PAZ-containing lobe of the bilobal Aa-Ago architecture. The first overhang nucleotide stacks over a tyrosine ring, while the second overhang nucleotide, together with the intervening sugar-phosphate backbone, inserts into a preformed surface cavity. Photochemical crosslinking studies on Aa-Ago with 5-iodoU-labeled single-stranded siRNA and siRNA duplex provide support for this externally bound siRNA-Aa-Ago complex. The structure and biochemical data together provide insights into a protein-RNA recognition event potentially associated with the RISC-loading pathway.
The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.
CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.
RNA interference (RNAi) is a new modality in gene therapy which can elicit down-regulation of gene expression and has enormous potential in the treatment of neurological diseases. RNAi is a conserved system through which double stranded RNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatus may be artificially triggered by delivery of naked siRNA molecules or by plasmid-based expression of dsRNA. Before these techniques can be used as effective treatments in the brain, efficient methods of in vivo delivery must be devised. This review first describes the mechanism of RNAi, and then critically examines both viral and non-viral methods for delivery of RNAi to the mammalian brain. There have been a number of important recent publications in this field and the progress towards effective in vivo delivery of RNAi to the central nervous system is discussed. Finally, potential problems that must be considered before applying this technology to the human brain are outlined.
In the past few years, our knowledge about small non-mRNAs (snmRNAs) has grown exponentially. Approaches including computational and experimental RNomics have led to a plethora of novel snmRNAs, especially small nucleolar RNAs (snoRNAs). Members of this RNA class guide modification of ribosomal and spliceosomal RNAs. Novel targets for snoRNAs were identified such as tRNAs and potentially mRNAs, and several snoRNAs were shown to be tissue-specifically expressed. In addition, previously unknown classes of snmRNAs have been discovered. MicroRNAs and small interfering RNAs of about 21-23 nt, were shown to regulate gene expression by binding to mRNAs via antisense elements. Regulation of gene expression is exerted by degradation of mRNAs or translational regulation. snmRNAs play a variety of roles during regulation of gene expression. Moreover, the function of some snmRNAs known for decades, has been finally elucidated. Many other RNAs were identified by RNomics studies lacking known sequence and structure motifs. Future challenges in the field of RNomics include identification of the novel snmRNA's biological roles in the cell.
Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.
OBJECTIVE: This paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression. METHODS: It was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR. RESULTS: It showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01). CONCLUSION: M1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.
SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58 875 004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing. AVAILABILITY AND IMPLEMENTATION: The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.