Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
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ABSTRACT:
PROVIDER: PRJNA682745 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13202014.fastq.gz | Fastqsanger.gz | |||
| SRR13202015.fastq.gz | Fastqsanger.gz | |||
| SRR13202016.fastq.gz | Fastqsanger.gz | |||
| SRR13202017.fastq.gz | Fastqsanger.gz | |||
| SRR13202018.fastq.gz | Fastqsanger.gz |
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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:Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.
| S-EPMC7818937 | biostudies-literature
Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress
Project description:Systematic Evaluation of crRNA Design Parameters for Optimized Cas13d-Mediated RNA Targeting in Chicken Cells
| PRJNA1307436 | ENA
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:CRISPR/Cas13 systems are increasingly becoming popular tools for programmable RNA targeting owing to its high-precision and ease at which crRNAs can be designed against mRNA targets of interest, often surpassing the usability of traditional RNA-targeting tools such as RNAi. The RNA-targeting capabilities of CRISPR/Cas13 have been harnessed to correct pathogenic mutations and combat infectious diseases. However, the wider adoption of Cas13 systems relies on the ability to design effective crRNAs that are broadly targetable and resistant to emerging mutants. In this study, we delineated the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for crRNAs, we designed multiple versions of crRNAs to investigate the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data revealed variable knockdown levels between crRNAs, in which a few crRNAs achieved over 95% target knockdown. We showed that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of the position at which the single-nt mismatch was introduced. However, 4-nt mismatches within crRNAs significantly reduced targeting efficacy, and eight nucleotide mismatches completely diminished targeting efficacy. Finally, we compared targeting efficiency of two widely used Cas13d variants (RfxCas13d and HfCas13d) and associated collateral degradation of bystander RNA. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.
2025-08-26 | GSE305666 | GEO
Project description:To examine the potential off-target effects by CRISPR/Cas13d-mediated HoxB13 knockdown in 293FT cells. To analyze differentially expressed genes upon CRISPR/Cas13d-mediated HoxB13 knockdown in LNCaP95 mouse liver metastasis.
2025-05-14 | GSE264062 | GEO
Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.
2014-11-24 | E-GEOD-62187 | biostudies-arrayexpress
Project description:To examine the potential off-target effects by CRISPR/Cas13d-mediated CTSL knockdown in 293FT cells
2022-06-09 | GSE203052 | GEO
Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels).
2014-11-24 | GSE62187 | GEO