Pervasive off-target activity in CRISPR-interference (CRISPRi) system [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1063363 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27483800.fastq.gz | Fastqsanger.gz | |||
| SRR27483801.fastq.gz | Fastqsanger.gz | |||
| SRR27483802.fastq.gz | Fastqsanger.gz | |||
| SRR27483803.fastq.gz | Fastqsanger.gz | |||
| SRR27483804.fastq.gz | Fastqsanger.gz |
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Project description:Pervasive off-target activity in CRISPR-interference (CRISPRi) system [ChIP-seq]
| PRJNA1063362 | ENA
Project description:CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.
2024-10-11 | GSE252977 | GEO
Project description:CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.
2024-10-11 | GSE252978 | GEO
Project description:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.
2022-06-30 | E-MTAB-8027 | biostudies-arrayexpress
Project description:In Mycobacterium tuberculosis, the efflux pump, EfpA, plays an essential but uncharacterised role in its physiology and antibiotic tolerance. In this study, an ATc-inducible CRISPR interference (CRISPRi) system was introduced into M. tuberculosis to knock-down the expression of efpA and determine any subsequent effects on the organism. As part of the study, microarray experiments were performed to determine any changes in gene expression caused by efpA repression M. tuberculosis by comparing the transcriptomic profile between ATc-induced efpA knock-down cultures to that of efpA knock-down cultures without ATc-induction.
2024-06-19 | E-MTAB-13755 | biostudies-arrayexpress
Project description:CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knockdown gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus. Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically-dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter. We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in Caulobacter.
2020-01-23 | GSE139521 | GEO
Project description:Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. To date, a genome-wide CRISPRi library, optimized for targeting the Saccharomyces cerevisiae genome, has not been presented. Here, we have generated a comprehensive, inducible CRISPRi library, based on spacer design rules optimized for yeast. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and low false discovery rate across a variety of experimental conditions, enabling rapid and reliable genome-wide assessment of genetic function and interactions in S. cerevisiae.
2020-11-27 | GSE159409 | GEO
Project description:Assess the on- and off-target effects of dox-inducible CRISPR/Cas9 and CRISPRi constructs in a human iPS cell line. Transcript quantification of 3 cell lines, each plus or minus doxycycline and with or without specific single guide RNAs (sgRNAs), with 2 biological replicates each.
2016-01-19 | E-GEOD-76980 | biostudies-arrayexpress
Project description:Gene expression in mammals is regulated by noncoding elements that can impact physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses CRISPR interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase (Mb) of sequence in the vicinity of 2 essential transcription factors, MYC and GATA1, and identify 9 distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the 7 enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer-promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.
2016-11-11 | GSE87257 | GEO
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.
2016-01-18 | E-GEOD-71490 | biostudies-arrayexpress