Ontology highlight
ABSTRACT:
PROVIDER: PRJNA911336 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22696405_1.fastq.gz | Fastqsanger.gz | |||
| SRR22696405_2.fastq.gz | Fastqsanger.gz | |||
| SRR22696406_1.fastq.gz | Fastqsanger.gz | |||
| SRR22696406_2.fastq.gz | Fastqsanger.gz | |||
| SRR22696407_1.fastq.gz | Fastqsanger.gz |
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Project description:To investigate the efficacy of CRISPR-Csm complexes for RNA- knockdown in eukaryotes, we quantified transcript abundance in HEK293T cells after targeting several nuclear or cytoplasmic RNAs. RNA-seq demonstrates efficient and specific knockdown of CRISPR-Csm compared to Cas13 and shRNA knockdown.
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Project description:RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a “proof of principle” demonstration that CRISPR endoribonuclease can be used for programmable mRNA transcript degradation in eukaryotes. Using zebrafish as the animal model and Csm complex as the CRISPR endoribonuclease, we targeted EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We also tested Csm mediated knockdown on the endogenous tdgf1 (oep) transcript. At optimal Csm dose, we observed a penetrance of the characteristic one-eyed phenotype at greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown with no significant off-target effects in our model.
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Project description:Transcriptome analysis of targeted RNA-knockdown using CRISPR-Csm in HEK293T cells
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Project description:CRISPR RNA isolated from the purified CSM complex from Sulfolobus solfataricus
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Project description:Purpose: This study aims to identify cell-subset specific methylated (CSM) loci in mammalian brain methylomes and to uncover epigenetic regulatory networks associated with brain development. Methods: To generate methylation data from DNA double strands simultaneously, genome-scale hairpin bisulfite sequencing technique was used for the assessment of the levels of asymmetric DNA methylation in the human frontal cortical gray and white matters Results and conclusion: CSM loci increase dramatically during early stages of brain development. They are overrepresented in cis-regulatory elements such as active enhancers and promoters, and are enriched for GWAS variants associated with neurological-disorder-related diseases/traits. Co-methylated CSM clustering analysis indicated that the CSMs hypo-methylated in neurons are enriched for EGR1 binding sites.Our results show the functional relevance of CSM loci identified and provide insights into epigenetic complexity of mammalian brains.
2017-02-14 | GSE70982 | GEO
Project description:Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we developed and methodically tested an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. Using the example gene RBFOX2, we validated protein-level changes and assessed the functional impact of isoform-specific knockdown. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all transcriptional and splicing events, including exon skipping and inclusion, alternative 5’ and 3’ splice sites, and alternative starts and ends.
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Project description:Consecutive sexual maturation (CSM), an abnormal reproductive phenomenon of a marine snail, Reishia clavigera, has occurred since 2017 in the vicinity of the Fukushima Daiichi Nuclear Power Plant after the nuclear disaster there. To determine the molecular basis for CSM, we conducted transcriptome profiling in the ganglia of normal and CSM snails.
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Project description:Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish
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Project description:Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, RNA-seq was used to identify both on-target and off-target binding sites for the synthetic TFs. The data in this submission were generated using the TALE transfection experiments.
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Project description:Mature mRNAs undergo quality control during translation that may lead to RNA degradation by triggering the nonsense mediated decay (NMD) pathway. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction or an intron in the 3'UTR activates NMD. However, many of the features that lead to the activation of this pathway are unclear. UPF1, an RNA helicase, is the core NMD factor. UPF1 forms a multi-protein complex by recruiting a series of factors and other protein complexes in a process that depends on the UPF1 phosphorylation-dephosphorylation cycle. Among the factors recruited by UPF1, SMG5-SMG7 and SMG6 have critical importance in executing NMD. The SMG5-SMG7 heterodimer induces the exonucleolytic degradation of the mRNA, which depends on the recruitment of deadenylation factors. SMG6 has endonucleolytic activity and cleaves the targeted transcript close to the stop codon. The redundancy between the exonucleolytic and endonucleolytic paths to achieve degradation during NMD has been previously reported in the literature. To investigate the apparent redundancy between SMG5-SMG7 and SMG6 activity and to further understand the features that lead to the activation of NMD, we have generated two clones of SMG7 knockout human cells using CRISPR-Cas9. We generated mRNA-Sequencing data for control and both SMG7 KO clones with additional siRNA-mediated knockdown of Luciferase (Luc) as control, SMG5 or SMG6.
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