Sequence-specific toxicity of dCas9 in bacteria
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ABSTRACT:
PROVIDER: PRJEB45922 | ENA |
REPOSITORIES: ENA
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Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition. five samples co-transfected with five uPA sgRNAs and each of the four dCas9 fusions, or control transfection with pUC19 plasmid
2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress
Project description:Over the last decade, gene-silencing mediated by dCas9 binding to transcribed regions or their promoters, a strategy referred to as CRISPRi, has emerged as a powerful tool in bacterial genetics. While this strategy has already been broadly adopted, several studies have reported experimental setups in which dCas9 expression was toxic. In particular, guide RNAs that share specific PAM-proximal sequence motifs were shown to be toxic to E. coli. Here we demonstrate that this toxicity is caused by off-target binding of dCas9 to the promoter of essential genes. Silencing of off-target genes can occur with as little as 4nt of identity between the PAM-proximal sequence and the off-target position. This phenomenon only occurs in some promoter sequences but does not appear to be constrained to any specific PAM-proximal sequence. Accordingly, screens performed in various strains of E. coli and related species shows that the nature of toxic guide RNAs changes together with the evolution of the sequence of off-target positions. These results highlight the importance of relying on several guide RNAs targeting the same gene when performing CRISPRi experiments in bacteria in order to avoid any possible confounding results due to off-target binding.
2022-10-25 | GSE216032 | GEO
Project description:Over the last decade, gene-silencing mediated by dCas9 binding to transcribed regions or their promoters, a strategy referred to as CRISPRi, has emerged as a powerful tool in bacterial genetics. While this strategy has already been broadly adopted, several studies have reported experimental setups in which dCas9 expression was toxic. In particular, guide RNAs that share specific PAM-proximal sequence motifs were shown to be toxic to E. coli. Here we demonstrate that this toxicity is caused by off-target binding of dCas9 to the promoter of essential genes. Silencing of off-target genes can occur with as little as 4nt of identity between the PAM-proximal sequence and the off-target position. This phenomenon only occurs in some promoter sequences but does not appear to be constrained to any specific PAM-proximal sequence. Accordingly, screens performed in various strains of E. coli and related species shows that the nature of toxic guide RNAs changes together with the evolution of the sequence of off-target positions. These results highlight the importance of relying on several guide RNAs targeting the same gene when performing CRISPRi experiments in bacteria in order to avoid any possible confounding results due to off-target binding.
2022-11-04 | GSE217004 | GEO
Project description:High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Our results also highlight the fact that off-targets with as little as nine nucleotides of homology to the guide RNA can strongly block gene expression. Altogether this study provides important design rules to safely use dCas9 in E. coli.
| S-EPMC5954155 | biostudies-other
Project description:Epigenetic aberration is one of the major driving factors in the initiation, promotion, and progression of human cancer and it is also often associated with acquired therapeutic resistance. Several chemical epigenetic modulators have been reported. However, results from animal models and clinical trials have indicated that severe on- and off-target toxicity is one of the leading factors behind failures in epidrug development. CRISPR/dCas9 technology provides a powerful tool for precise epigenetic regulation at the single-gene level. This approach requires ectopic expression of exogenous epigenetic modulatory proteins, which cause side effects and technical challenges for preparation and delivery. Recently, we have shown that Cas9 tagging with the peptide motif (FCPF) was recognized by perfluoro biphenyl derivatives. Here, we introduced the FCPF-tag into dCas9 and report a chemically induced platform for epigenome editing (Chem-CRISPR/dCas9FCPF). By conjugating JQ1, a BRD4 inhibitor, to perfluoro biphenyl (JQ1-FCPF), we demonstrate that JQ1-FCPF covalently binds to CRISPR/dCas9 and specifically inhibits BRD4 in proximity to the promoters/enhancers of c-MYC with the guidance of c-MYC sgRNAs. Thus, we achieved epigenetic modulation of c-MYC with high efficiency and specificity.
2023-06-09 | GSE232132 | GEO
Project description:ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE
| PRJNA387472 | ENA
Project description:Tracking of dCas9-methyltransferase footprints
| PRJNA347354 | ENA
Project description:dCas9 activation of RP11-326A19.4
| PRJNA548121 | ENA
Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition.
2016-06-01 | GSE74935 | GEO
Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
2020-05-01 | GSE129397 | GEO