Browse
Submit Data
Databases
API
Help

Dataset Information

32 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

RNA virus interference via CRISPR/Cas13a system in plants.

ABSTRACT: CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

SUBMITTER: Aman R

PROVIDER: S-EPMC5755456 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

RNA virus interference via CRISPR/Cas13a system in plants.

Aman Rashid R Ali Zahir Z Butt Haroon H Mahas Ahmed A Aljedaani Fatimah F Khan Muhammad Zuhaib MZ Ding Shouwei S Mahfouz Magdy M

Genome biology 20180104 1

<h4>Background</h4>CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.<h4>Results</h4>CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expre ...[more]

PMID: 29301551

Similar Datasets

CRISPR-Cas13d mediates robust RNA virus interference in plants.

Project description:BackgroundCRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana.ResultsOur data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta.ConclusionsOur data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

| S-EPMC6886189 | biostudies-literature

Universal theranostic CRISPR/Cas13a RNA-editing system for glioma.

Project description:Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses. Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.

| S-EPMC10614676 | biostudies-literature

The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Project description:Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

| S-EPMC10994916 | biostudies-literature

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection.

Project description:Background & aimsHepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR-Cas13a technology.MethodsWe established fluorescence (F) and lateral flow strip (L) assays based on CRISPR-Cas13a combined with RT-PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR-Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT-qPCR and RT-ddPCR.ResultsFor synthetic HDV RNA plasmids, the sensitivity of RT-PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT-qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT-qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT-qPCR, RT-ddPCR, RT-PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively.ConclusionsWe developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

| S-EPMC10796118 | biostudies-literature

Establishing RNA virus resistance in plants by harnessing CRISPR immune system.

Project description:Recently, CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR-Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR-Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus-targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.

| S-EPMC6041442 | biostudies-literature

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes.

Project description:In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.

| S-EPMC7064736 | biostudies-literature

The CRISPR-Cas13a Gene-Editing System Induces Collateral Cleavage of RNA in Glioma Cells.

Project description:RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR-Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR-Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA-EGFP induces a "collateral effect" after knocking down the target gene in EGFP-expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR-Cas13a system induces death in EGFRvIII-overexpressing glioma cells. Bulk and single-cell RNA sequencing analysis in U87-Cas13a-EGFRvIII cells confirm the collateral effect of the CRISPR-Cas13a system. Furthermore, CRISPR-Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR-Cas13a system in cancer cells and the powerful tumor-eliminating potential of this system.

| S-EPMC6794629 | biostudies-literature

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.

Project description:In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.

| S-EPMC6125684 | biostudies-literature

CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

Project description:Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

| S-EPMC8219879 | biostudies-literature

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

Project description:The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

| S-EPMC6619288 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data