Functional genomic screens in human cells to identify host factors for SARS-CoV-2 and common cold coronaviruses
Ontology highlight
ABSTRACT: We performed genome-wide CRISPR KO screens in human Huh7.5.1 cells to select for mutations that render host cells resistant to viral infection by SARS-CoV-2, human coronavirus 229E and OC43.
Project description:Rituximab (RTX) is widely used as a first-line therapeutic strategy for patients affected by immune thrombocytopenia (ITP). However, a large proportion of patients relapse after successful treatment. The present NGS assay was done to help find the cause for this relapse on the immune repertoire level. Therefore, we performed antibody repertoire sequencing for three RTX relapse patients with subsequent mutation and clonal analysis, as well as for two patients with ongoing ITP and two healthy donors (HD) with subsequent mutation analysis.
Project description:To identify the B cell subset(s) involved in T-independent (TI) responses in humans, we vaccinated healthy individuals with Pneumovax, a model TI vaccine composed of 23 capsular polysaccharides (capPS). High-throughput repertoire sequencing of day 7-plasma cells (PC-d7) and of different B cell subpopulations before and after vaccination was done to search for clonal filiations between anti-capPS PC-d7 clones and the isolated B cell subsets. Ig-Rep seq was done for PC-d7 of 6 vaccinee, and for different subsets sampled at day 0, 7, 14, 28 and 56 from 3 vacinees.
Project description:Biological replicates of liver spheroids derived from primary hepatocytes were treated with IFNa2 and/or baricitinib for 24 h and subsequently infected with Sars-CoV-2. The cells were harvested 48 h time after infection. Total RNA was extracted and sequenced with a strand-specific paired end RNA-seq protocol.
Project description:The immunosuppressive tumour microenvironment constitutes a significant hurdle to immune checkpoint inhibitors response. Both soluble factors and specialised immune cells such as regulatory T cells (TReg) are key components of active intratumoural immunosuppression. Previous studies have shown that Inducible Co-Stimulatory receptor (ICOS) can be highly expressed in the tumour microenvironment, especially on immunosuppressive TReg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from paired tumour and peripheral blood of 5 patients with non-small cell lung cancer (NSCLC). We found Icos mRNA expression in NSCLC samples was higher in TRegs than in other T cell subsets and higher in the TME than in the periphery with the expression pattern in both compartments following the general trend of: TReg > CD4non-TReg > CD8 > Other.
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:We sought to investigate the extent of genetic and antigen-driven predetermination of antibody repertoires throughout B-cell development (pre-B cells, naive B cells, plasma cells). To this end, we used (i) an inbred model organism (mouse, C57BL/6J) to quantify genetic predetermination and (ii) cohort-stratification (4 cohorts: 1 untreated, 3 antigen-immunized) to quantify antigen-driven predetermination. To ensure generalizability of our research, we additionally sequenced naive B cells from Balb/c and pet shop mice.
Project description:Huh7.5.1 cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, Huh7.5.1 cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the druggable genome library at MOI 0.3. Cells were then selected with puromycin, expanded, and then pooled together and cryofrozen in aliquots. Cells were thawed constituting over 1000× genome coverage worth of mutagenized library. The cells were infecting with DENV2_429557 and DENV-2_16681_Hap1-adapted at MOI of 0.1. Virus-resistant colonies were harvested. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.
Project description:To test the differences in genome-wide DNA methylation signatures of haploid, diploid and triploid hESCs, we extracted genomic DNA from these cells and performed RRBS.
Project description:To search for factors regulating paternally imprinted genes (PEGs), we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs, and further analyzed the molecular phenotype upon perturbation of candidate PEGs regulators.
Project description:Members of the apicomplexans are defined by apical cytoskeletal structures and secretory or-ganelles, tailored for motility and invasion. Gliding is powered by the actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In Toxoplasma gondii, the conoid, composed of a cone of spiraling tubulin fibers and apposed preconoidal rings (PCRs), is an enigmatic, dynamic organelle of undefined function. Here we mapped five new components of the PCRs and deduce that the structure serves as a pivotal hub for actin polymerization and glideosome assembly. F-actin produced by Formin1 on the PCRs is used by Myosin H to generate the force for conoid extrusion. A set of conserved B-box-type zinc finger domain containing proteins in Apicomplexa is indispensable for PCRs formation, co-noid extrusion and motility in Toxoplasma and Plasmodium. Conoid dynamics directs the flux of F-actin to the pellicular space, acting as dynamic gatekeeper to tightly control parasite motility during invasion and egress.