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Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA and of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

ABSTRACT: Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA and of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

PROVIDER: PRJNA797093 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17612600_1.fastq.gz	Fastqsanger.gz
	SRR17612600_2.fastq.gz	Fastqsanger.gz
	SRR17612601_1.fastq.gz	Fastqsanger.gz
	SRR17612601_2.fastq.gz	Fastqsanger.gz
	SRR17612602_1.fastq.gz	Fastqsanger.gz

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Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA

Project description:To determine whether target genes were specifically knocked down by RfxCas13d in N2a cells, we performed RNA-seq to compare the differentially expressed genes between cells transfected with targeting crRNAs and non-targeting crRNA.

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Next-generation sequencing facilitates transcriptomes quantitative analysis of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

Project description:To determine what effect the collateral activity of RfxCas13d has on cells, a HEK293T cell line stably expressing RfxCas13d (HEK293T-RfxCas13d) was constructed and then transfected with plasmids encoding target gene and corresponding crRNAs. After 24 hours of transfection, cells were collected and extracted for total RNA. Then, we performed RNA-seq to compare the differentially expressed genes between cells transfected with targeting crRNAs and non-targeting crRNA.

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Next Generation Sequencing Facilitates Transcriptomes Quantitative Analysis of HEK293T cells transfected with the plasmids respectively encoding RfxCas13d, crRNA and NeuN.

Project description:To determine what effect the collateral activity of RfxCas13d has on HEK293T cells upon targeting overexpressed NeuN. The plasmids encoding RfxCas13d, NeuN and NT/targeting crRNA into HEK293T cells. After 24 hours of transfection, cells were collected and extracted for total RNA. Then, we performed RNA-seq to compare the differentially expressed genes between cells transfected with targeting crRNAs and non-targeting crRNA.

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Next Generation Sequencing Facilitates Transcriptomes Quantitative Analysis of N2A cells transfected with the plasmids respectively encoding RfxCas13d/dRfxCas13d and crRNA.

Project description:To determine what effect the collateral activity of RfxCas13d has on N2acells. The plasmids encoding RfxCas13d/dRfxCas13d and crRNA were transfected into N2a cells. After 48 hours of transfection, GFP-positive cells were collected using FCAS and extracted for total RNA. Then, we analyzed the transcriptome of each group with RNA-seq and analyzed the differential genes among the groups.

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Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA

Project description:Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA

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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.

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Next-generation sequencing facilitates transcriptomes quantitative analysis of N2a cells transfected with RfxCas13d and targeting/non-targeting crRNA and of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-02-10 | GSE193668 | GEO

Next-generation sequencing facilitates transcriptomes quantitative analysis of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

Project description:Next-generation sequencing facilitates transcriptomes quantitative analysis of stably expressing RfxCas13d HEK293T cells transfected with target genes and corresponding crRNAs

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