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ABSTRACT: mRNA sequencing to assess CasRx and DjCas13d specificity in HEK293FT

PROVIDER: PRJNA857683 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20078700_1.fastq.gz	Fastqsanger.gz
	SRR20078700_2.fastq.gz	Fastqsanger.gz
	SRR20078701_1.fastq.gz	Fastqsanger.gz
	SRR20078701_2.fastq.gz	Fastqsanger.gz
	SRR20078702_1.fastq.gz	Fastqsanger.gz

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Project description:mRNA sequencing to assess CasRx and DjCas13d specificity in hESC (H1)

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Cas13bt3 knockdown of VEGFA mRNA in mammalian cells and mouse retina [RNA-Seq]

Project description:Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme for anti-VEGFA therapy. Methods: Mammalian cells (HEK293FT) or mouse retina were treated with shRNA, CasRx or Cas13bt3 carrying VEGFA targeting sgRNA. Total RNA was collected for RNA seqeuencing to study VEGFA mRNA expression and well as off-target genes. Results: Significant knockdown of VEGFA mRNA was observed with no effect on predicted off-target genes in mammalina cells and mouse retina. No loss of mouse VEGFA was also detected. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA.

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mRNA sequencing to assess RfxCasR and matching shRNA specificity

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Whole transcriptomes analyses of HEK293FT cell line treated with ISLR secreted supernatant or empty vector control supernatant

Project description:Purpose: To explore the molecular mechanisms of ISLR protein, RNA sequencing was performed to analyze the geome-wide change of ISLR secreted supernatant treated HEK293FT cells compared to those of control supernatant treated cells. Methods: Total mRNA was extracted from HEK293FT cells in triplicate respectively. Then RNA quality was assessed using an Agilent Bioanalyzer 2100 and the sample reads were sequenced using Illumina Hiseq 4000 platform. As a reasult, we got the transcript data using Hisat2 followed by Stringtie. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: We mapped about 60 million sequence reads per sample to the human genome and identified about 58,000 transcripts in HEK293FT cells. Many transcripts showed different expression between ISLR secreted supernatant and control treated HEK293FT cells, with a fold change ≥2 and p value <0.05. Gene set enrichment analysis (GSEA) showed a significantly positive enrichment of Hippo target genes. Conclusion: Our study present the datiled transcripts analysis of HEK293FT cells, with biologic replicates. Based on RNA-seq transcriptome characterization , we conclude a molecular mechanism of ISLR regulating multiple pathways related to tissue repair.

2020-03-20 | GSE135280 | GEO

RNA-Targeting CRISPR/CasRx System Relieves Disease Symptoms in Huntington's Disease Models

Project description:Huntington's disease (HD) is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (HTT) gene. Recent advances in gene editing technologies, such as CRISPR/CasRx, have opened new avenues for therapeutic interventions. In this study, we explored the efficacy of CRISPR/CasRx, which can specifically and accurately digest single-stranded RNA and down-regulate the expression of related genes, in targeting the HTT mRNA and its potential as a treatment strategy for HD. Our results showed that CRISPR/CasRx could significantly down-regulate HTT mRNA in different models, including human embryonic kidney (HEK) 293T cells, HD140Q-knockin (HD 140Q-KI) mice at various disease stages, and Huntingtin knockin (HD-KI) pigs, and lead to a subsequent decrease in the expression of mutant Huntingtin (mHTT) protein. Moreover, this intervention could significantly ameliorate the neurological symptoms in HD 140Q-KI mice and HD-KI pigs. These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.

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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

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Project description:Deep Sequencing of mRNA from Drosophila melanogaster, Drosophila Sechellia, and their F1 hybrid. These data were generated in a study to analyze the extent and specificity of trans-splicing in Drosophila. mRNA-seq libraies were prepared from poly(A)+ RNA prepared from whole F1 hybrids of D. melanogaster and D. sechellia (female 0-3d post eclosion). Separate control libraries were prepared from D. melanogaster and D. sechellia total RNA (mixed before library preparation). The results of this study identified 80 novel trans-splicing events between homologous alleles of the same gene. Keywords: RNA-Seq Keywords: Expression profiling by high throughput sequencing There were 2 total samples in this study. Paired-end mRNA-seq was used to survey trans-splicing in F1 hybrids of D. melanogaster and D. sechellia. A mixed "control" library was used to assess the frequency of false-positive trans-splicing signal resulting from errors in reference genomes, high-throughput sequencing, and RT-PCR amplification based strand-switching.

2010-07-19 | E-GEOD-22588 | biostudies-arrayexpress

CRISPR activation of long non-coding RNAs transiently expressed during cortical neuron differentiation associated with Field, et al, Stem Cell Reports 2018

Project description:4 transiently expressed long non-coding RNAs that were identified in human and non-human primate cortical organoid differentiation were activated out of context in HEK293FT cells using CRISPRa.

2018-10-04 | GSE120702 | GEO

E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [RNA-seq]

Project description:The purpose of this experiment was to characterize MazF cleavage specificity and locations of cleavage genome-wide in E. coli and also identify any leaderless transcripts .To do this, MazF was expressed in a ΔmazF background and compared to an empty vector. rRNA-subtracted samples were used to assess cleavage in the mRNA, and total RNA samples were used to assess changes in the rRNA.

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Identification of the molecular basis of anti-cancer effect of Huaier.

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