Genome-wide CRISPR-Cas9 screen identifies protein-coding genes and microRNAs important in FLT3-ITD+ AML (MV4-11) cellular growth
ABSTRACT: The functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to test the impact of individual microRNAs on the growth of FLT3-ITD positive leukemia cells. This approach identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote tumor cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. Our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease. Loss-of-function CRISPR-Cas9 screen identifies genes whose loss leads to increased or decreased FLT3-ITD+ cell growth over 23 day time-course
Project description:The expression profile in miR-155-/- FLT3-ITD+ AML is unknown. Using empty vector (EV) or two distinct miR-155 (S3 or S10) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing to determine the expression profile in these cells dependent on miR-155. We found a number of pathways dysregulated, including STAT5 activation. RNAseq was performed on EV or miR-155 lentiviral CRISPR-Cas9 infected MV4-11 cell lines in triplicate cultures.
Project description:The expression level of microRNAs in FLT3-ITD+ AML is unknown. Using empty vector (EV) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing on small RNAs to determine microRNA expression level in these cells. We found a variety of evolutionarily conserved and non-conserved microRNAs expressed in our cells of interest. Small RNAseq on EV lentiviral CRISPR-Cas9 infected MV4-11 cell lines was performed on triplicate cultures.
Project description:We generated these data to compare two modifications of the original ATAC-seq protocol. One was the cleavage of mtDNA using CRISPR/Cas9 and 100 gRNAs targeting mtDNA. The other was the removal of detergent from the cell lysis step. There are 27 sample pairs, untreated and treated with anti-mt CRISPR/Cas9 grouped by sample pair number. Refer to Supplemental File 1 of the article describing this data set for more information on the samples.
Project description:One of the strongest associated type 2 diabetes (T2D) loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. To aid in the identification of the actual gene(s) under the influence of this variant, we first generated a CRISPR/Cas9 mediated 1.4kb deletion of the genomic region harboring rs7903146 in the HCT116 cell line followed by global gene expression analysis. HCT116 cells with or without a CRISPR/Cas9 mediated1.4kb deletion of the genomic region harboring the SNP rs7903146 were analyzed for expression, with 3 replicates per condition (DEL vs WT). We observed 99 genes with significant differential expression (FDR cut-off=10%) and an effect size of at least two-fold. We then carried out high-throughput chromosome conformation capture assays in the HCT116 and NCM460 cell lines and in colon tissue (see experiment E-MTAB-4845) in order to ascertain which of these perturbed genes’ promoters made consistent physical contact with the genomic region harboring the variant. This revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2.
Project description:We introduce the Multiplexed Editing Regulatory Assay (MERA), a high-throughput CRISPR/Cas9-based approach that analyzes the regulatory genome for function in its native context. By tiling thousands of mutations across ~40 kb of cis-regulatory genomic space and using knock-in GFP reporters to read out gene activity, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. In addition, we performed a deep-sequencing strategy to find basepair-resolution functional motifs involved in regulation of the gene by sequencing thousands of functional and non-functional genotypes at genomic locations perturbed by specific guide RNAs. Design of 3908 gRNAs to perturb regulatory regions associated with the genes Tdgf1,Nanog,Zfp42 and Rpp25 as well as 10 GFP-targetting positive controls. Also, deep-sequencing of genomic regions mutated by 6 selected gRNAs after sorting the electroporated cells into GFP-positive and GFP-negative populations.
Project description:Activation of JAK-STAT3 signaling by leukemia inhibitory factor (LIF) is required for maintaining self-renewal of mouse embryonic stem cells (mESCs). STAT3 perform cell type-specific roles in different cell type, here we revisit the role of STAT3 using mouse female germ stem cell (mFGSCs). We applied CRISPR/Cas9 system to generate Stat3 knockout FGSCs and then observed cell growth inhibition and cell cycle arrest in KO cell line. By combining genome wide ChIP-Seq and RNA-Seq, we identified 5990 STAT3 binding sites and discovered serval genes specific regulated by STAT3 that were involved in stem cell proliferation and female gonad development in FGSCs. In general, we identify key roles of STAT3 for sustains self-renewal and proliferation for FGSCs in this study.
Project description:The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context- dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. Additional data can be found at tko.ccbr.utoronto.ca RNAseq of five human cell lines with Cas9 knock-ins.
Project description:We performed a large-scale genome-wide characterisation of indels generated following editing with CRISPR/Cas9. We used pools of sgRNAs and performed targeted capture and sequencing of the edited regions in HepG2 cells.
Project description:One of the strongest associated type 2 diabetes (T2D) loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. To aid in the identification of the actual gene(s) under the influence of this variant, we first generated a CRISPR/Cas9 mediated 1.4kb deletion of the genomic region harboring rs7903146 in the HCT116 cell line followed by global gene expression analysis (see experiment E-MTAB-4839). We then carried out high-throughput chromosome conformation capture assays in the HCT116 and NCM460 cell lines and in colon tissue in order to ascertain which of these perturbed genes promoters made consistent physical contact with the genomic region harboring the variant. To assess consistency and reproducibility we utilized two different techniques: Circularized Chromosome Conformation Capture (4C) and Capture C. In both types of assays, after preparing 3C libraries, our bait of interest was the region harboring rs7903146. Loci interacting with such bait are enriched for by inverse PCR in 4C and by oligonucleotide capture in capture C. 4C assays were carried out in the following samples: two on NCM460 cells (using different primer sets), one on HCT116 cells, one on HCT116 cells with a CRISPR/Cas9 mediated 1.4kb deletion of the genomic region harboring the SNP rs7903146, and one on colon tissue. Capture C assays were carried out in one sample each of the cell lines NCM 460 and HCT116. This experiment, coupled with the associated E-MTAB-4839, revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2.
Project description:We sought to characterise how chromatin states affected CRISPR/Cas9 activity and the induced indel profiles. We used two different doses of Trichostatin and two different doses of a EZH2 inhibitor to modulate the chromatin state and then performed targeted DNA-seq of selected sgRNA target regions to identify the indels.