Dataset Information

Proteome of Ins1E beta cell line with CerS2 ablation

ABSTRACT: To gain insight into changes of the proteome of the rat beta cell line Ins1E with and without ablation of CerS2, shotgun proteomics was performed in control cells (transfected withCas9 plasmid without gRNA) and CerS2 KO cells (transfected with Cas9 plasmids with 2 gRNAs, ablating most of the coding exons). Ablation of CerS2 was confirmed by PCR, qPCR and Western Blot.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Pancreatic Beta Cell Line, Pancreatic Endocrine Cell

SUBMITTER: Bengt-Frederik Belgardt

LAB HEAD: Bengt-Frederik Belgardt

PROVIDER: PXD029848 | Pride | 2022-09-22

REPOSITORIES: Pride

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			Action	DRS
	20072018_C2Tha_ctrl-3h_6598_DIA28.raw	Raw
	20072018_C2Tha_ctrl-3h_6606_DIA28.raw	Raw
	20072018_C2Tha_ctrl_3h_6602_DIA28.raw	Raw
	20072018_C2Tha_ko_3h_6600_DIA28.raw	Raw
	20072018_C2Tha_ko_3h_6604_DIA28.raw	Raw

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Project description:Background: The CRISPR/Cas9 toolbox has recently been expanded to include approaches for modulating gene expression. To successfully build on this work, and apply it for answering biological questions, it is important to establish it in a broad range of circumstances. Genome-scale CRISPR interference (CRISPRi) has been used in human cells lines, however the rules for designing effective guide RNAs (gRNAs) in different organisms are not well known. We sought to determine rules that determine gRNA effectiveness at transcriptional repression in Saccharomyces cerevisiae. Results: We created an inducible single plasmid CRISPRi system for gene repression in yeast, and used it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identified previously-described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments allowed us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility were clearly more effective. We also found the best region to target gRNAs was between the transcription start site (TSS) and 200bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, point mutations were tolerated equally well by truncated (18 nt specificity sequence) and full length (20 nt) gRNAs, however, 18 nt gRNAs were generally less potent than full length gRNAs. Conclusions: Our results establish a powerful functional genomics screening method, provide rules for designing effective gRNAs for gene repression, and show that 18 nt and 20 nt gRNAs exhibit similar tolerance to mismatches in the target sequence. These findings will enable effective library design and genome-wide screening in many genetic backgrounds. An expression construct was created for inducible CRISPRi in yeast. Key features include ORFs expressing dCas9-Mxi1 and the tetracycline repressor (TetR), as well as a tetracycline inducible gRNA locus containing the RPR1 promoter with a TetO site, a NotI site for cloning new gRNA specificity sequences, and the constant part of the gRNA. When yeast containing this plasmid are grown in the absence of anhydrotetracycline (ATc) TetR binds the gRNA promoter and prevents PolIII from binding and transcribing the gRNA. This in turn prevents dCas9-Mxi1 from binding the target site. In the presence of ATc, TetR dissociates and gRNA is expressed, allowing dCas9-Mxi1 to bind its target locus, and repress gene expression. gRNA libraries were cloned into this construct and transformed into yeast to create pools. Experiments were conducted in which yeast pools were grown in inducing (+ATc) and non-inducing conditions (-ATc) in the presence of different drugs. After multiple generations of growth in these conditions, yeast plasmids were minipreped and the gRNA locus was PCRed and sequenced via MiSeq. Counts of each gRNA were compared in different conditions.

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Proteome of Ins1E beta cell line with CerS2 ablation

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