Chemically synthesized guide RNAs can direct CRISPR-CasRx cleavage of circRNAs with high efficiency and specificity
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ABSTRACT:
PROVIDER: PRJNA785254 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17085613.fastq.gz | Fastqsanger.gz | |||
| SRR17085614.fastq.gz | Fastqsanger.gz | |||
| SRR17085615.fastq.gz | Fastqsanger.gz | |||
| SRR17085616.fastq.gz | Fastqsanger.gz | |||
| SRR17085617.fastq.gz | Fastqsanger.gz |
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Project description:In this study, we demonstrate that CRISPR/RfxCas13d (CasRx)-mediated circRNA depletion is, for the circRNAs studied, more efficient than Argonaute 2-dependent short hairpin RNA (agoshRNA)-mediated depletion and with fewer off-target effects on the linear host RNAs. We also designed and optimally used synthetic guide RNAs (syn-gRNAs) in combination with CasRx to efficienctly deplete circRNA ciRS-7. None of the knockdown strategies tested (pre-gRNA, gRNA, syn-gRNA and agoshRNA) showed any significant off-target effects at the transcriptomics level. Differential expression analysis was performed for each KD setup versus non-targeting control (three biological replicates of each). In conclusion, CasRx-mediated circRNA knockdown using both vector-based and syn-gRNA strategies is a useful tool for future studies on circRNA functions.
2023-04-30 | GSE189963 | GEO
Project description:To explore the overall circRNAs involved in growth and development of Arabidopsis thaliana across the lifespan, we deeply sequenced samples of whole plants from different developmental stages (cotyledons emergence, rosette leavesï¹¥1 mm, rosette growth complete, first flower open, flourishing florescence, first silique shattered, senescence). The total RNA was purified by rRNA-depletion and linear RNA removal with RNAseR, and sequenced by the Illumina HiSeq2500 platform. We obtained 31 Gb raw data and identified 1217 circRNAs with expression quantity. We annotated these circRNAs and predicted their targeted microRNA. The circRNAs involved in growth and development of Arabidopsis thaliana across lifespan were identified and analyzed using the Illumina HiSeq2500 platform.
2016-09-01 | E-GEOD-77672 | biostudies-arrayexpress
Project description:Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
2020-12-06 | GSE162720 | GEO
Project description:Synthesized DNA with unnatural nucleotides Synthetic Genomics
| PRJNA89669 | ENA
Project description:CRISPR-Cas13 systems have recently been employed to silence RNAs in yeast, plants, mammalian cell lines and animals, however, collateral effects induced by Cas13 may impede their research and therapeutic applications. Here, we found Cas13a and CasRx-mediated RNA knockdown induced substantial collateral effects on both exogenous and endogenous genes in mammalian cells. Moreover, transgenic mice carrying CasRx, with or without targeting guide RNA, exhibited high lethality. Therefore, our results present limitations of CasRx, requiring a solution to address its fidelity before clinical applications.
2025-09-02 | GSE157586 | GEO
Project description:Background: Of Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), 85%-90% of cases develop from liver cirrhosis, and circular RNAs (circRNAs) have important roles in this process; however, differences in serum circRNA expression profiles between patients with HBV-related cirrhosis and those with HCC have not been studied. Methods: Serum RNA was extracted from patients with HCC and cirrhosis (n = 5 per group) and used for microarray analysis of circRNA expression profiles. Bioinformatics analyses, including clustering, differential expression, and construction of a ceRNA network, were performed. Quantitative real-time reverse transcription PCR validation analysis was conducted using samples from patients with HBV-related cirrhosis (n = 88) and HCC (n = 73). Further, statistical analyses were used to analyze the potential function and value of selected circRNAs with expression differing between the HBV-related cirrhosis and HCC groups. Results: Cluster analysis revealed 8 up-regulated and 80 down-regulated circRNAs. Further, qRT-PCR analysis showed that circRNA_0000367 expression was consistent with that detected by microarray experiments, with significantly lower levels in patients with HBV-related HCC than those with HBV-related cirrhosis. CircRNA_0000367 expression levels were also significantly lower in patients with drug-resistant HBV and those with HBV-related cirrhosis with Model for End-Stage Liver Disease score < 10. Further, circRNA_00000367 expression levels were lower in patients with HBV-related cirrhosis who progressed to HCC. Analysis of the lncRNA-miRNA-mRNA ceRNA network identified 39 miRNAs and 24 mRNAs involved in circRNA_00000367 networks; these target genes were involved in various biological processes and signaling pathways. Conclusion: Serum cicrRNA_0000367 is a potential HCC biomarker in patients with HBV-related cirrhosis, where down-regulation of cicrRNA_0000367 in HBV-related cirrhosis may be associated with progression to HCC.
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Project description:Chemically-induced osteogenic cells (ciOG)
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Project description:The early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combination of signalling pathways at play remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (<96 hours) and efficiently (80%) guide embryoid bodies to form a three dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered by parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epi in vitro. Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.
2021-04-01 | GSE129655 | GEO
Project description:Transcriptome-wide investigation of circRNAs in rice.
| PRJEB8082 | ENA
Project description:Intensive research in past two decades has uncovered the presence and importance of noncoding RNAs (ncRNAs), which includes microRNAs (miRs) and long ncRNAs (lncRNAs). These two classes of ncRNAs interact to a certain extent, as some lncRNAs bind to miRs to sequester them. Such lncRNAs are collectively called 'competing endogenous RNAs' or 'miRNA sponges'. In this study, we screened for lncRNAs that may act as miRNA sponges using the publicly available data sets and databases. To uncover the roles of miRNA sponges, loss-of-function experiments were conducted, which revealed the biological roles as miRNA sponges. LINC00324 is important for the cell survival by binding to miR-615-5p leading to the de-repression of its target BTG2 LOC400043 controls several biological functions via sequestering miR-28-3p and miR-96-5p, thereby changing the expressions of transcriptional regulators. Finally, we also screened for circular RNAs (circRNAs) that may function as miRNA sponges. The results were negative at least for the selected circRNAs in this study. In conclusion, miRNA sponges can be identified by applying a series of bioinformatics techniques and validated with biological experiments. The expressions of miRs were examined in HEK-293 cells that were LINC00324- or LOC400043-silenced using siRNA.
2016-07-06 | E-GEOD-73689 | biostudies-arrayexpress