Project description:<p>Mass spectrometry–based proteomic analysis of an osteocyte line, MLO-Y4, was conducted to profile global protein expression changes under two experimental settings, 1) CRISPR-Cas9-based knockout of CFTR versus wild-type; 2) Cl- deprivation versus normal extracellular medium.</p>
Project description:Since LAMC2 is a secreted molecule present in the extracellular matrix of the cells, was designed a strategy based on CRISPR/Cas9-mediated homologous recombination to mark LAMC2 cells in human PDACs. A Cas9 single-guide RNAs complementary to sequences overlapping the stop codon of the LAMC2 locus was designed and a donor vector that contained LAMC2 homology arms flanking an EGFP reporter cassette positioned immediately upstream of the stop codon was generated. LF2A self-cleavage peptide in frame with EGFP so that LAMC2-EGFP locus was expressed as a single mRNA was added, whereas the resulting polypeptide was cleaved in the two encoded proteins, LAMC2 and EGFP. L3.6pl and PANC-1 cells were nucleofected with the donor vector together with a guide-RNA-Cas9 (guide). The engineered cells were subcutaneously injected in CD1 male mice and RNA-seq was performed on LAMC2-EGFP+ and EGFP- -derived tumors.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:In the present study, Marchantia polymorpha Mppcs loss of function mutants were generated through CRISPR/cas9 mediated genome-editing. To assess whether the knockout of MpPCS gene affects the transcription of M. polymorpha nuclear genes in unstressed condition, the Mppcs-2 knockout mutant and Cam2 wild-type transcriptomes were compared by RNA-Seq.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:We performed RNA-seq analysis on Spp1 wild-type and CRISPR/Cas9-mediated Spp1 knock out pancreatic tumour organiod from Pdx1-Flp;KrasFSF-G12D;Trp53frt/frt; Vim-EGFP (KPFV) mice. We compared the transcriptome between these two groups of them with the aim to understand their molecular signatures and the relevance to human pancreatic ductal adenocarcinoma subtypes.
Project description:A validation experiment performed on HEK293 cell lines after genome editing. The design contains three duplicate runs consisted of: HEK293 wild type cell line HEK293 with MIR484 gene knockdown using CRISPR-Cas9 HEK293 with MIR185 gene knockout using CRISPR-Cas9
Project description:Dysfunction of blood vessels leads to severe vasculature pathogenesis. Previous studies have demonstrated that constitutive NFkB activation results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NFkB regulates blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into human vascular derivatives to study how NFkB modulates vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating vascular inflammation, survival, vasculogenesis, differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon TNFa stimuli. Lastly, further evaluation of gene expression patterns in IkBa knockout vascular cells demonstrated that IkBa acted largely independent of NFkB signaling pathway. Taken together, our data reveals a protective role of NFkB/RelA in modulating human blood vessel homeostasis and maps the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.
Project description:CHD8, encoding Chromodomain helicase DNA binding protein 8, is a top autism spectrum disorders (ASDs) risk gene. To better understanding the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to mimic the loss of function status that would exist in the developing human embryo prior to neuronal differentiation. Transcriptome profiling (RNA-seq) in neural progenitors and early differentiating neurons revealed that CHD8 hemizygosity (CHD8+/-) affected the expression of several thousands of genes, enriched for functions of neural development, β-catenin/Wnt signaling, extracellular matrix, and skeletal system development. Moreover, CHD8 regulates multiple genes implicated in ASD, schizophrenia and genes associated with brain volume. iPSCs derived from a healthy subject were transduced with CRISPR/Cas9 vectors with single guide RNA sequences to target the N-terminal of CHD8 protein to generate truncated mutation seach of the two target sequences. Two clones, one with a 2-bp (KO1) and the other with a 10-bp (KO2) heterozygous deletion were found.The CHD8+/- iPSC lines were used to generate NPCs and early differentiating neurons for RNA-seq analysis, together with samples prepared from the parental clones, for a total of 8 samples (two biological replicates of wild-type (WT) and CHD8+/- at two neurodevelopmental stages).
Project description:Cells were transduced with a genome-wide CRISPR knockout library to introduce gene disruptions across the genome. After Cas9-induced DNA double-strand breaks, repair outcomes were captured and quantified using high-throughput sequencing. This experiment aims to identify genetic factors influencing specific DNA repair outcome patterns such as 1-bp insertions, microhomology-mediated deletions, and large insertions.