Project description:The Mobile CRISPRi system with and without mRFP-targeting sgRNA was engineered into Pseudomonas aeruginosa PA14 strain with chromosomally encoded mRFP. RNA was isolated from these strains, and the corresponding cDNA library was synthesized and sequenced in 150 bp paired-end reads. Approximately 1,000,000 reads were collected for each of the two samples, with ~94% alignment to PA14 WT by Bowtie254, and transcripts were counted with HTSeq55. Only genes with a non-normalized read count greater than 1 in both samples were included in analysis, with a coverage of 1286 genes (~20% genome). This data shows that the Mobile CRISPRi system is selective for sgRNA-guided knockdown of mRFP.
Project description:Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.
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.
Project description:Small RNAs recently emerged as a new class of mobile instructive signals in development. Here, we investigate their mechanism of action and show that the gradients formed by mobile small RNAs generate sharply defined domains of target gene expression. By modulating the source of artificial miRNAs we show that boundary formation is an inherent property of the small RNA gradient itself. The threshold-based readout of such gradients is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. In addition to generating sharp expression domains of their immediate targets, the readouts of opposing small RNA gradients enable formation of stable and uniformly positioned developmental boundaries. These novel patterning properties of small RNAs are reminiscent of those of morphogens in animal systems. However, their exceptionally high specificity, direct mode of action, and the fully intrinsic nature of their gradients, distinguish mobile small RNAs from classical morphogens. Our findings present mobile small RNAs and their targets as highly portable and evolutionarily-tractable regulatory modules through which to create pattern in development and beyond.
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.
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.