Hepatitis Delta Virus Ribozyme Research Articles

Rapid Rescue of Goose Astrovirus Genome via Red/ET Assembly.

The host-specific infection of Avian Astrovirus (AAstVs) has posed significant challenges to the poultry industry, resulting in substantial economic losses. However, few reports exist on the functional consequences of genome diversity, cross-species infectivity and mechanisms governing virus replication of AAstVs, making it difficult to develop measures to control astrovirus transmission. Reverse genetics technique can be used to study the function of viruses at the molecular level, as well as investigating pathogenic mechanisms and guide vaccine development and disease treatment. Herein, the reverse genetics technique of goose astrovirus GAstV/JS2019 strain was developed based on use of a reconstructed vector including CMV promotor, hammerhead ribozyme (HamRz), hepatitis delta virus ribozyme (HdvRz), and SV40 tail, then the cloned viral genome fragments were connected using Red/ET recombineering. The recombinant rGAstV-JS2019 was readily rescued by transfected the infectious clone plasmid into LMH cells. Importantly, the rescued rGAstV/JS2019 exhibited similar growth kinetics comparable to those of the parental GAstV/JS2019 isolate in cultured cells. Our research results provide an alternative and more effective reverse genetic tool for a detailed understanding of viral replication, pathogenic mechanisms, and molecular mechanisms of evolution.

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling five cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis.

Ribonucleases and small nucleolytic ribozymes are both able to catalyze RNA strand cleavage through 2'-O-transphosphorylation, provoking the question of whether protein and RNA enzymes facilitate mechanisms that pass through the same or distinct transition states. Here, we report the primary and secondary 18O kinetic isotope effects for hepatitis delta virus ribozyme catalysis that reveal a dissociative, metaphosphate-like transition state in stark contrast to the late, associative transition states observed for reactions catalyzed by specific base, Zn2+ ions, or ribonuclease A. This new information provides evidence for a discrete ribozyme active site design that modulates the RNA cleavage pathway to pass through an altered transition state.

The synthesis of Broccoli RNA fluorescent aptamer in <i>Saccharomyces cerevisiae</i> yeast cells

BACKGROUND: RNA aptamers are short, single-stranded oligonucleotides, with remarkable binding ability to target molecules characterized by high specificity and affinity. Such targets are vastly diverse and range from specific ions to entire cells. RNA aptamers are widely used in biology and medicine for basic research, as well as for practical purposes as in therapy and diagnostics. At present, chemical or in vitro methods of synthesis are mainly used to obtain RNA aptamers. However, such methods are expensive and time-consuming with low productivity. Therefore, in vivo methods are becoming more attractive to researchers working on optimizing high-scale production of RNA aptamers.
 AIM: The aim of this work is to develop a reporter system for optimizing the synthesis of small RNA molecules in Saccharomyces cerevisiae yeast cells.
 MATERIALS AND METHODS: We used the Broccoli fluorescent RNA aptamer to develop a reporter system allowing us to optimize the conditions for in vivo short RNA synthesis in yeast cells. This aptamer is about 112 bp in size and binds to the fluorogenic dye DFHBI-1T. Only upon binding, the aptamer-dye complex exhibits fluorescence properties. After excitation using light with a wavelength of 482 nm, the aptamer-dye complex emission is observed with a peak at 505 nm.
 RESULTS: We have designed a reporter system providing the synthesis of the fluorescent Broccoli RNA aptamer in S. cerevisiae yeast cells. Transcription of RNA molecules containing the aptamer is carried out by the regulated promoter of the GAL1 gene. The synthesized transcripts contain the HH and HDV ribozymes to ensure precise cleavage of the RNA aptamer sequences.
 CONCLUSIONS: This reporter system is based on the Broccoli RNA aptamer, and it can be used to optimize the in vivo synthesis of RNA aptamers in S. cerevisiae yeast cells. This work serves an urgent task in connection with the active use of such aptamers in scientific research, biotechnology and medicine.

Identification of selective hepatitis delta virus ribozyme inhibitors by high-throughput screening of small molecule libraries

Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and hepatocellular carcinoma. Hepatitis delta virus (HDV)'s tight dependency on hepatitis B virus and the host cell machinery for its life cycle limits the development of direct-acting antivirals. Thus, we aimed to identify compounds that could block HDV replication by targeting its antigenomic ribozyme. We generated stable Huh7 human hepatoma cells expressing a reporter gene (Gaussia luciferase) either downstream (Gluc-2xRz) or upstream (2xRz-Gluc) of two HDV antigenomic ribozyme sequences. We performed high-throughput screening of three small molecule libraries. The secreted luciferase was measured as a readout of ribozyme inhibition upon addition of the molecules. Each plate was considered valid when the Z factor was >0.4. Specificity and toxicity evaluations were performed for the hits with a Z-score >5 and half-maximal inhibitory concentration was calculated by performing a dose-response experiment. A dose-dependent induction of luciferase expression was detected in Gluc-2xRz-transfected cells incubated with the antisense morpholino, suggesting that the catalytic activity of the ribozyme cloned downstream of the reporter gene was efficiently inhibited. Among the 6,644 compounds screened, we identified four compounds that showed a specific inhibitory effect on the HDV antigenomic ribozyme in Gluc-2xRz cells, i.e. three histone deacetylase inhibitors and the purine analogue 8-azaguanine. The latter also significantly decreased HDV replication (by 40%) in differentiated HepaRG cells six days post infection. Using a novel cell culture model, we identified four small molecules active against the antigenomic HDV ribozyme. These results may provide insights into the structural requirements of molecules designed for the potent and specific inhibition of HDV replication. Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and the development of hepatocellular carcinoma. Despite the current development of several new compounds, there is still a need for efficient antiviral treatments specifically targeting hepatitis delta virus (HDV). This work describes a novel cell culture model that allows for the high-throughput screening of compounds able to inhibit HDV ribozymes. We identified four small molecules active against the antigenomic HDV ribozyme (the ribozyme involved in the early step of HDV replication), with the strongest activity shown by 8-azaguanine, a purine analogue. Our data may provide insights into the structural requirements of molecules designed to inhibit HDV.

Generation of a DNA-launched classical swine fever virus infectious clone packaged in bacterial artificial chromosome

Reverse genetics system offers powerful tool for the research of RNA viruses. The infectious clones of classical swine fever virus (CSFV) were commonly constructed either in high- or low-copy number plasmids and transcribed to infectious RNA using phage RNA-polymerases. Herein, the full-length genome of CSFV Shimen strain, flanked by cytomegalovirus immediate-early (CMV) promoter (a eukaryotic RNA polymerase II promoter) sequence at the 5′-end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3′-end, was packaged in bacterial artificial chromosome vector to establish a CSFV infectious clone pBAC-smCSFV. This infectious cDNA clone maintained stability after passaged 20 times in bacteria. Transfection of PK15 cells with this cDNA clone facilitated recovery of infectious progeny virus which was identical to parent virus as characterized by RT-qPCR, western blotting, indirect immunofluorescence assay, one-step growth kinetics analysis and nucleotide sequencing. Based on this CSFV infectious cDNA clone, the mCherry was inserted between viral Npro and C protein to develop reporter virus CSFV-mCherry. The mCherry was stably expressed after CSFV-mCherry was passaged 10 times in PK15 cells. Taken together, this present study develops a concise and efficient CSFV infectious cDNA clone and a reporter virus CSFV-mCherry. To the best of our knowledge, this is the first combination of CMV promoter and BAC system in construction of CSFV reverse genetics system. The CSFV infectious cDNA clone and the reporter virus will be useful in the study of CSFV virus biology, virulence determinants, molecular pathogenesis, vaccine development and virus-host interaction.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

Inspecting the Ribozyme Region of Hepatitis Delta Virus Genotype 1: Conservation and Variability.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688–771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715–745. No difference in QS was observed over time. The most variable region was between nt 739–769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Development and Characterization of a Reverse Genetics System for a Human-Derived Severe Fever With Thrombocytopenia Syndrome Virus Isolate From South Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2.

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

Synthesis of Oligoribonucleotides Containing a 2'-Amino-5'-S-phosphorothiolate Linkage.

Oligoribonucleotides containing a photocaged 2′-amino-5′-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2′-amino-5′-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2′-amino-5′-S-phosphorothiolate linkage. We incorporated the photocaged 2′-amino-5′-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate’s nucleophilic 2′-NH2 or a poor fit of the modified transition state at the ribozyme’s active site.

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a "plateau" in the pH-rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL-rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Infectious Clones of Tomato Chlorosis Virus: Toward Increasing Efficiency by Introducing the Hepatitis Delta Virus Ribozyme.

Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3′ end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3′ ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones.

Construction of an infectious clone of Zika virus stably expressing an EGFP marker in a eukaryotic expression system

BackgroundZika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP.MethodsA PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot.ResultsRescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy.ConclusionOur results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

Efficient multiplex genome editing by CRISPR/Cas9 in common wheat.

Common wheat has a large genome with three subgenomes (A, B and D), making it challenging to create mutations at multiple genomic sites simultaneously. The CRISPR/Cas9 system offers a game changing tool for editing crop genomes (Chen et al., 2019). Three main strategies have been developed to produce multiple single guide RNAs (sgRNAs), including the conventional multiplex system with tandem repeats of separate U3 or U6 promoters (TRSP), the tRNA processing system (Xie et al., 2015), and the ribozyme processing system (Gao and Zhao, 2014).

SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast.

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pomberely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1promoter. To overcome these problems we have developed an improved system, SpEDIT, thatuses a synthesised Cas9 sequencecodon-optimised for S. pombeexpressed from the medium strength adh15promoter.The SpEDITsystem exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence.Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDITsystem allowed a 100% mutagenesis efficiency to be achieved when generating targeted pointmutants in the ade6 + or ura4 +genes by transformation of cells from asynchronous cultures. SpEDITalso permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDITsystem displayed reduced toxicity compared to currently available S. pombeediting systems. Thus, SpEDITprovides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

Design and Evaluation of AgoshRNAs with 3'-Terminal HDV Ribozymes to Enhance the Silencing Activity.

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18base pairs (bp) stem and a 3-5 nucleotides(nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.