Project description:A knockout cell library in Huh7.5.1 cells was generated by introducing a genome-scale CRISPR library (GeCKOv2, Addgene #1000000049) and subjected to hepatitis A virus infection (HM175/18f) to isolate virus-resistant mutant cells. Genomic DNA was isolated from the original and virus-selected mutant cell populations and abundance of guideRNA encoding sequences were measured by sequencing on an Illumina NextSeq (High Output).
Project description:Data of gene expression levels across individuals, cell types, and disease states is rapidly expanding, yet we have limited understanding of how expression levels impact cellular and organismal phenotypes. Here, we present a massively parallel system for assaying the effect of gene expression levels on cellular fitness in Saccharomyces cerevisiae by systematically altering the expression level of each of ~100 endogenous genes at ~100 distinct expression levels spanning a 500-fold range at high resolution. Our results show that the relationship between expression levels and growth is gene- and environment-specific, with the specific relationship exhibited by each gene being highly informative on its function, stoichiometry within complexes, and interaction with other genes. Notably, in one of the two environmental conditions that we tested, we find that ~20% of the genes have expression levels where fitness is greater than that at wild-type expression levels, indicating that wild-type expression is not optimal for growth in that condition. We find that genes whose fitness is greatly affected by small changes in expression level tend to exhibit lower cell-to-cell variability in expression, suggesting that noise in gene expression is shaped in part by the relationship between expression and fitness. Overall, our study addresses a fundamental gap in our understanding of the functional significance of gene expression regulation and offers a powerful framework for evaluating the phenotypic effects of expression variation. 130 synthetic promoters were genomically integrated upstream of 96 endogenous yeast genes to span an expression range for each gene. Fitness as a function of the expression level of each gene was computed by a pooled growth competition assay.
Project description:A375 and HT1080 cells are treated with G-quadruplex ligands PDS or PhenDC3 following genome-wide shRNA knockdown. This enables the identification of genes that when silenced, specifically compromises cell growth in the presence of the ligand. First, a pilot screen was performed to determine a ligand concentration and experimental duration that caused ligand-specific, significant changes in shRNA levels. Second, a genome-wide screen was performed to globally evaluate G4-ligand synthetic lethal interactions. Third, to corroborate the G4-sensitisers uncovered in the genome-wide screen, a focussed screen was performed with a custom shRNA pool.
Project description:To identify new therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 "knockout" (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Tyr15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. A whole-genome CRISPR-Cas9 knockout screens targeting over 18,000 genes using the all-in-one LV-sgRNA:Cas9 platform system were performed using a “shot gun” approach by transducing 2 GBM patient-derived isolates and 2 human neural stem cell isolates with the pool library (2 biological replicates), and cultures were outgrown for ~3 weeks. The end time point of each screen was compared to day 0 in order to determine which sgRNAs were overrepresented or underrepresented in the population.
Project description:Here we developed CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starr-seq technique. Extensive assessment of CapStarr-seq demonstrated accurate quantification of enhancer activity. Furthermore, we found that enhancer strength correlates with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. CapStarr-seq analysis in P5424 cell line (2 replicates), 3T3 cell line and in the plasmid library before (Input) and after transfection
Project description:Huh-7.5.1 cells were treated with 0.2% DMSO, 20 microM NeoB for 24 h. Treatment with 0.2% DMSO for 24h was prepared as non-treated Huh7.5.1 cells. Huh7.5.1 cells were kindly provided by Prof. Francis Chisari at The Scripps Research Institute. Total RNA obtained from NeoB-treated and un-treated Huh7.5.1 cells
Project description:Comparsion of DNA interacting proteome of BC muants to assess chnages in transcriptional proteins. Four strain comparsion between WT vs delta pglL vs delta ogc vs delta pglL complemented AmrAB::S7-pglL-his. LFQ based quantification
Project description:In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-related processes. For P. falciparum, the causative agent of human malaria, the nucleosome landscape of the extremely AT-rich intergenic regulatory regions is largely unexplored. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit. We observe strong positioning of nucleosomes around splice sites that could aid co-transcriptional splicing events. In addition, nucleosome depleted regions are apparent hallmarks of transcription start sites (TSSs) and may support pre-initiation complex assembly. Moreover, we reveal nucleosome occupancy dynamics on strong TSSs during intraerythrocytic development, which correlate with gene expression changes and we observe a characteristic nucleosome architecture of functional - but not inert - TGCATGCA DNA motifs. Collectively, these findings highlight the regulatory capacity of the nucleosome landscape of this deadly human pathogen. Mnase-seq during the intra-erythrocytic asexual cycle of Plasmodium falciparum var2csa-panned 3D7 parasites for 8 time-points, every 5 hours starting from 5 hours post invasion until 40 hours post-invasion (T5-T40). Cycle length of these parasites is ~43 hours, synchronicity window is ~ 8 hours. T40 has 2 technical replicates (independent digestions; T40A, T40B). Additionally, pellet control sample (T15), histone H4-ChIP control (T40A) and sonicated and amplified genomic DNA. Chromatin was digested using a combined MNase + exonuclease III treatment. Libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification. Strand-specific RNA-seq for expression quantification during the intra-erythrocytic asexual cycle of Plasmodium falciparum var2csa-panned 3D7 parasites for 8 time-points every 5 hours starting from 5 hours post invasion invasion until 40 hours post-invasion (T5-T40). Cycle length of these parasites is ~43 hours, synchronicity window is ~ 8 hours. These samples are originating from the exact same batch of parasites as are the MNase-Seq libraries. Libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.