Tool For Gene Silencing Research Articles

A novel gene silencing strategy based on tobacco rattle virus in Hibiscus mutabilis.

Hibiscus mutabilis L. is a popular regional characteristic plant in China, cultivated for its attractive flower colors, extended bloom time, and medicinal properties. To enhance molecular breeding and gene function studies, we conducted transcriptome analysis and identified valuable genes in previous research. Nonetheless, the current inefficient and labor-intensive transformation techniques have hindered their applications. Virus-induced gene silencing (VIGS) provides a precise and effective strategy for post-transcriptional down-regulation of endogenous gene expression. We investigated the performance of tobacco rattle virus (TRV) as a tool for targeting and silencing the gene encoding the protein involved in chloroplast development, cloroplastos alterados 1 (altered chloroplast; CLA1), of H. mutabilis through Agrobacterium tumefaciens-mediated infiltration. By effectively suppressing the CLA1 gene associated with chloroplast development in H. mutabilis via the TRV-VIGS system, we have illustrated the inaugural implementation of VIGS in this species. Quantitative RT-PCR proved that HmCLA1 expression in agro-infiltrated plants was lower than in the mock-infiltrated (mock) and the control (CK) plants. Phenotypic observations corroborated the albino phenotype in leaves following successful HmCLA1 silencing. Our study showcases TRV-VIGS as a potential gene silencing tool for H. mutabilis, facilitating functional genomics studies and molecular breeding efforts in this species.

Bacterial outer membrane vesicle nanorobot

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Patient-Derived Exosomes as siRNA Carriers in Ovarian Cancer Treatment.

RNA interference is a powerful gene-silencing tool with potential clinical applications. However, its therapeutic use is challenging because suitable carriers are unavailable. Exosomes are stable small endogenous vesicles that can transport functional molecules to target cells, making them ideal small interfering RNA (siRNA) carriers. Herein, we elucidated the therapeutic potential of patient-derived exosomes as an siRNA carrier for ovarian cancer (OC) treatment. The exosomes were extracted from the culture medium of primary fibroblasts collected from the omentum of patients with OC during surgery. MET proto-oncogene, receptor tyrosine kinase (MET) was selected for gene silencing, c-Met siRNAs were synthesized and loaded into the exosomes (Met-siExosomes) via electroporation, and the treatment effect of the Met-siExosomes was assessed in vitro and in vivo. The Met-siExosomes downregulated the c-Met protein levels and inhibited OC cell proliferation, migration, and invasion. In xenograft experiments using SKOV3-13 and ES-2 cells, Met-siExosomes were selectively extracted from peritoneally disseminated tumors. Intraperitoneal treatment suppressed the c-Met downstream targets in cancer cells and prolonged mouse survival. The synthesized siRNAs were successfully and selectively delivered via the exosomes to intraperitoneally disseminated tumors. As patients with OC routinely undergo omentectomy and abundant fibroblasts can be easily collected from the omentum, patient-derived exosomes may represent a promising therapeutic siRNA carrier to treat OC.

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development.

Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

DCas13-mediated translational repression for accurate gene silencing in mammalian cells

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

Reduction of UreB and CagA expression level by siRNA construct in Helicobacter pylori strain SS1

BackgroundTwo important virulence factors, urease and cagA, play an important role in Helicobacter pylori (H. pylori) gastric cancer. Aim of this study was to investigate the expression level and function of ureB and cagA using small interfering RNAs (siRNA).MethodsSS1 strain of H. pylori was considered as host for natural transformation. siRNA designed for ureB and cagA genes were inserted in pGPU6/GFP/Neo siRNA plasmid vector to evaluate using phenotypic and genotypic approaches. Then, qPCR was performed for determining inhibition rate of ureB and cagA gene expression.ResultsThe expression levels of siRNA-ureB and siRNA-cagA in the recombinant strain SS1 were reduced by about 5000 and 1000 fold, respectively, compared to the native H. pylori strain SS1. Also, preliminary evaluation of siRNA-ureB in vitro showed inhibition of urea enzyme activity. These data suggest that siRNA may be a powerful new tool for gene silencing in vitro, and for the development of RNAi-based anti-H. pylori therapies.ConclusionOur results show that targeting ureB and cagA genes with siRNA seems to be a new strategy to inhibit urease enzyme activity, reduce inflammation and colonization rate.

Distinct pathways for export of silencing RNA in Caenorhabditis elegans systemic RNAi

Dietary supplied double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) systemically in some animals, including the nematode Caenorhabditis elegans. Although this phenomenon has been utilized as a major tool for gene silencing in C.elegans, how cells spread the silencing RNA throughout the organism is largely unknown. Here, we identify two novel systemic RNAi-related factors, REXD-1 and TBC-3, and show that these two factors together with SID-5 act redundantly to promote systemic spreading of dsRNA. Animals that are defective in all REXD-1, TBC-3, and SID-5 functions show strong deficiency in export of dsRNA from intestinal cells, whereas cellular uptake and processing of dsRNA and general secretion events other than dsRNA secretion are still functional in the triple mutant animals. Our findings reveal pathways that specifically regulate the export of dsRNA in parallel, implying the importance of spreading RNA molecules for intercellular communication in organisms.

An oral dsRNA delivery system based on chitosan induces G protein-coupled receptor kinase 2 gene silencing for Apolygus lucorum control

RNA interference (RNAi) is recognized as a new and environmentally friendly pest control strategy due to its high specificity. However, the RNAi efficiency is relatively low in many sucking insect pests, such as Apolygus lucorum. Therefore, there is an urgent need to develop new and effective ways of dsRNA delivery. Bacterially expressed or T7 synthesized dsRNA targeting a G Protein-Coupled Receptor Kinase 2 gene was mixed with chitosan in a 1:2 ratio by mass. The size of the chitosan/dsRNA nanoparticles was 69 ± 12 nm, and the TEM and AFM images showed typical spherical or ellipsoidal structures. The chitosan nanoparticles protected the dsRNA from nuclease activity, and pH and temperature-dependent degradation, and the fluorescently-tagged nanoparticles were found to be stable on the surface of green bean plants (48 h) (Phaseolus vulgaris) and were absorbed by midgut epithelial cells and transported to hemolymph. Once fed to the A. lucorum nymph, chitosan/dsRNA could effectively inhibit the expression of the G protein-coupled receptor kinase 2 gene (70%), and led to significantly increase mortality (50%), reduced weight (26.54%) and a prolonged developmental period (8.04%). The feeding-based and chitosan-mediated dsRNA delivery method could be a new strategy for A. lucorum management, providing an effective tool for gene silencing of piercing-sucking insects.

Superparamagnetic iron oxide nanoparticles target BxPC-3 cells and silence MUC4 for the treatment of pancreatic cancer

PurposeSuperparamagnetic iron oxide nanoparticles (SPION) are excellent magnetic resonance imaging (MRI) contrast agents. Mucin 4 (MUC4) acts as pancreatic cancer (PC) tumor antigen and influences PC progression. Small interfering RNAs (siRNAs) are used as a gene-silencing tool to treat a variety of diseases. MethodsWe designed a therapeutic probe based on polyetherimide-superparamagnetic iron oxide nanoparticles (PEI-SPION) combined with siRNA nanoprobes (PEI-SPION-siRNA) to assess the contrast in MRI. The biocompatibility of the nanocomposite, and silencing of MUC4 were characterized and evaluated. ResultsThe prepared molecular probe had a particle size of 61.7 ± 18.5 nm and a surface of 46.7 ± 0.8mV and showed good biocompatibility in vitro and T2 relaxation efficiency. It can also load and protect siRNA. PEI-SPION-siRNA showed a good silencing effect on MUC4. ConclusionPEI-SPION-siRNA may be beneficial as a novel theranostic tool for PC.

Gold nanoparticles (AuNPs) can rapidly deliver artificial microRNA (AmiRNA)-ATG6 to silence ATG6 expression in Arabidopsis.

We establish a fast and efficient transient silencing system that facilitates functional studies of some genes, whose knockout leads to plant lethality. In plants, the generation of loss-of-function mutants is crucial for studying gene function. Artificial microRNA (AmiRNA) technology is a more targeted and effective tool for gene silencing. Gold nanoparticles (AuNPs) can bind nucleic acids and deliver them into animal cells. Here, AuNPs are used in combination with AmiRNA technology in plants. We found that AmiRNA-autophagy-related proteins (ATG6) can be delivered to cells by AuNPs to achieve the effect of ATG6 silencing. It is worth noting that on the 10th day there is still a silencing effect. Similar to the atg5 lines, silencing of ATG6 significantly reduced plant resistance to Pseudomonas syringae pv.maculicola (Psm) ES4326/AvrRpt2. Interestingly, ATG6 silencing and ATG5 mutation in NPR1-GFP (nonexpressor of pathogenesis-related genes) lines significantly reduced plant resistance to Psm ES4326/AvrRpt2, suggesting that autophagy is also involved in NPR1-regulated plant immune responses. In summary, we establish a fast and efficient transient silencing system that facilitates functional studies of some genes, whose knockout leads to plant lethality.

Neutrophil-Membrane-Coated Biomineralized Metal-Organic Framework Nanoparticles for Atherosclerosis Treatment by Targeting Gene Silencing.

Antisense oligonucleotides (ASOs) are promising tools for gene silencing and have been exploited as therapeutics for human disease. However, delivery of therapeutic ASOs to diseased tissues or cells and subsequent escape from the endosomes and release of ASO in the cytosol remain a challenge. Here, we reported a neutrophil-membrane-coated zeolitic imidazolate framework-8 (ZIF-8) nanodelivery platform (AM@ZIF@NM) for the targeted transportation of ASOs against microRNA-155 (anti-miRNA-155) to the endothelial cells in atherosclerotic lesions. Neutrophil membrane could improve plaque endothelial cells targeting through the interaction between neutrophil membrane protein CD18 and endothelial cell membrane protein intercellular adhesion molecule-1 (ICAM-1). The ZIF-8 "core" provided high loading capacity and efficient endolysosomal escaping ability. Delivery of anti-miR-155 effectively downregulated miR-155 expression and also saved the expression of its target gene BCL6. Moreover, RELA expression and the expression of its downstream target genes CCL2 and ICAM-1 were correspondingly reduced. Consequently, this anti-miR-155 nanotherapy can inhibit the inflammation of atherosclerotic lesions and alleviate atherosclerosis. Our study shows that the designed biomimetic nanodelivery system has great application prospects in the treatment of other chronic diseases.

Allosteric Nucleic Acid Enzyme: A Versatile Stimuli-Responsive Tool for Molecular Computing and Biosensing Nanodevices.

Allostery is a naturally occurring mechanism in which effector binding induces the modulation and fine control of a related biomolecule function. Deoxyribozyme (DNAzyme) with catalytic activity and substrate recognition ability isideal to be regulated by allosteric strategies. However, the current regulations frequently confront various obstacles, such as severe activity decay, signal leakage, and limited effectors. In this work, a rational regulation strategy for developing versatile effectors-responsive allosteric nucleic acid enzyme (ANAzyme) by introducing an allosteric domain in response to diverse effectors is established. The enzyme-like activity of this re-engineered ANAzyme can be modulated in a more predictable and fine way compared with the previous DNAzyme regulation strategies. Based on the allosteric strategy, the construction of allosterically coregulatory nanodevices and a series of basic logic gates and logic circuits are achieved, demonstrating that the proposed ANAzyme-regulated strategy showed great potential in molecular computing. Given these facts, the rational design of ANAzyme with the allosteric domain presented here can expand the available toolbox to develop a variety of stimuli-responsive allosteric DNA materials, including molecular machines, computing systems, biosensing platforms, and gene-silencing tools.

Liver-directed drugs for transthyretin-mediated amyloidosis.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis.

RNAi knockdown of CAPAr gene affects survival and fecundity of Bemisia tabaci (Gennadius)

Graphical abstract representing the functioning of CAPA receptor in Bemisia tabaci diuresis process and silencing of CAPAr resulting into inactivation of diuresis. • CAPAr gene silencing resulted into significant whitefly mortality. • Reduced fecundity and survival of CAPAr knockdown whiteflies. • Reduced relative gene expression of CAPAr in dsRNA fed whiteflies. Bemisia tabaci (Gennadius) Asia II 1 cause significant economic losses to cotton crop, directly by feeding on sap and indirectly through virus transmission. Although the current control methods are the combination of different tactics but still the chemical control is heavily relied. B. tabaci has gained resistance to majority of the insecticides which has made it difficult to manage this pest using synthetic chemicals. RNAi based gene silencing has been evolved as potential biochemical tool for specific gene silencing of target insect-pest and has been successfully demonstrated for Hemipteran insects. Here, we report the successful silencing of the CAPAr gene in B. tabaci which encodes for the G-protein coupled receptor (a transmembrane membrane peptide). Silencing of CAPAr gene resulted into reduced survival and fecundity of B. tabaci . Oral delivery of dsRNA mediated artificial diet corresponding to CAPAr gene resulted into significantly high adult mortality of 30.74% in treatment dsCAPAr 1.0 µg/µl after 48 hr of dsRNA feeding. A significant reduction in whitefly female fecundity was recorded for all the dsCAPAr concentrations with lowest value of 53.51 eggs per female in dsCAPAr 1.0 µg/µl. The relative gene expression of CAPAr gene further confirms the down regulation of CAPAr gene in knock down adult whiteflies. Our investigation proves the importance of CAPAr for B. tabaci survival and may be exploited as potential tool which can be strategic in integrated management programme for B. tabaci .

Abstract 4000: ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines

Abstract Approximately 50% of metastatic melanomas possess wild-type (WT) BRAF alleles. These BRAF-WT tumors are a significant clinical problem, despite the fact that these tumors commonly possess mutations in NF1 or RAS genes, as there are no targeted therapeutics for these tumors. Here we demonstrate that ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines, suggesting that disrupting ERBB4 signaling may be effective against BRAF WT melanomas. ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are validated targets in a variety of solid tumors, whereas the roles that ERBB4 plays in human malignancies are ambiguous. Our previous in silico analyses of BRAF-WT tumor genomes in The Cancer Genome Atlas skin cutaneous melanoma (TCGA-SKCM) dataset suggest that ERBB4 mutation or overexpression cooperates with NF1 or RAS gene mutations to drive melanomas via activation of the PI3 kinase/AKT signaling pathway. These in silico analyses have also enabled us to prioritize the ERBB4 mutants found in BRAF-WT melanomas. We have tested this hypothesis using a panel of human BRAF-WT melanoma cell lines. We have shown that expression of wild-type ERBB4 causes increased clonogenic proliferation, whereas expression of a dominant-negative ERBB4 mutant (K751M) causes decreased clonogenic proliferation. These data indicate that ERBB4 is sufficient and necessary for clonogenic proliferation of BRAF-WT melanoma cell lines. Analgous studies are testing whether ERBB4 regulates anchorage independence in semi-solid medium and proliferation rates and saturation density in culture dishes. The BRAF-WT melanoma cell lines endogenously express ERBB4 ligands, EGFR, and ERBB2. Moreover, ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR and ERBB4-ERBB2 heterodimers are oncogenic. Thus, we are using gene silencing and pharmacologic tools to determine whether endogenous ERBB4 ligands, endogenous EGFR, or endogenous ERBB2 is required for the oncogenic activity of ERBB4. Furthermore, we are determining whether prioritized ERBB4 mutants found in BRAF-WT melanomas exhibit greater signaling and oncogenic activities than WT ERBB4. Consequently, these experiments will establish a functional role for ERBB4 mutations and expression in BRAF-WT melanomas and will identify potential strategies for disrupting oncogenic ERBB4 signaling in these tumors. Citation Format: Lauren M. Lucas, Vipasha Dwivedi, Rania H. Mohamedelhassan, Erin L. Harrell, Connor M. Kelley, Elizabeth L. Knerr, Jessica A. Markham, David J. Riese. ERBB4 is sufficient and necessary for proliferation of BRAF WT melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4000.

Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes.

Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for P. thermosuccinogenes DSM 5809. By overcoming restriction barriers, transformation efficiencies of 102 CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered E. coli HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in P. thermosuccinogenes, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain’s fermentation profile. Although the details of the functioning of the restriction modification systems require further study, in vivo methylation can already be applied to improve transformation efficiency of P. thermosuccinogenes. Making use of the ThermodCas9-based CRISPRi, this is the first example demonstrating that genetic engineering in P. thermosuccinogenes is feasible and establishing the way for metabolic engineering of this bacterium.

The Effect of Dicer Knockout on RNA Interference Using Various Dicer Substrate Small Interfering RNA (DsiRNA) Structures.

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease Dicer. Dicer-substrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with an increased effectiveness of up to 100-fold compared to traditional 21-mer designs. DsiRNAs have a novel asymmetric design that allows them to be processed by Dicer into the desired conventional siRNAs. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood. In this study, to gain a deeper understanding of Dicer function in DsiRNAs, we designed nicked DsiRNAs with and without tetra-loops to target a specific mRNA sequence, established a Dicer knockout in the HCT116 cell line, and analyzed the efficacy of various DsiRNAs on RNAi-mediated gene silencing activity. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. We demonstrated that tetra-looped DsiRNAs exhibited increased efficacy for gene silencing, which was mediated by Dicer protein. Thus, this study improves our understanding of Dicer function, a key component of RNAi silencing, which will inform RNAi research and applications.

Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics Screens and Analysis of Gene Function in Cannabis sativa L.

Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis.

Making sense of the natural antisense transcript puzzle.

In plants, thousands of genes are associated with antisense transcription, which often produces noncoding RNAs. Although widespread, sense-antisense pairs have been implicated in a limited variety of functions in plants and are often thought to form extensive dsRNA stretches triggering gene silencing. In this opinion, we show that evidence does not support gene silencing as a major role for antisense transcription. In fact, it is more likely that antisense transcripts play diverse functions in gene regulation. We propose a general framework for the initial functional dissection of antisense transcripts, suggesting testable hypotheses relying on an experiment-based decision tree. By moving beyond the gene silencing paradigm, we argue that a broad and diverse role for natural antisense transcription will emerge.

RISC in Entamoeba histolytica: Identification of a Protein-Protein Interaction Network for the RNA Interference Pathway in a Deep-Branching Eukaryote.

ABSTRACTEntamoeba histolytica is a protozoan parasite that causes amebiasis in humans and is a major health concern in developing countries. Our previous work revealed a functional RNA interference (RNAi) pathway in Entamoeba. Several unusual features encompass the RNAi pathway in the parasite, including small RNAs (sRNAs) with a 5′-polyphosphate structure (identified to date only in Entamoeba and nematodes) and the conspicuous absence of a canonical Dicer enzyme. Currently, little is known about the Entamoeba RNA-induced silencing complex (RISC), which is critical in understanding how RNAi is achieved in the parasite. In this study, we examined the RISC of EhAgo2-2, the most highly expressed Argonaute protein in Entamoeba. We identified 43 protein components of EhAgo2-2 RISC with a broad range of functional activities. Two proteins with nucleosome assembly protein (NAP) domains, not previously observed in other RNAi systems, were identified as novel core members of amebic RISC. We further demonstrated the interaction of these NAPs with Ago using an in vitro recombinant system. Finally, we characterized the interaction network of five RISC components identified in this study to further elucidate the interactions of these RNAi pathway proteins. Our data suggest the presence of closely interacting protein groups within RISC and allowed us to build a map of protein-protein interactions in relation to Ago. Our work is the first to elucidate RISC components in Entamoeba and expands the current knowledge of RISC to a deep-branching single-celled eukaryote.