Genotyping of C.elegans mutants - CRISPR/Cas9 of all GPCR and neuropeptide genes
Ontology highlight
ABSTRACT: CRISPR/Cas9 genome editing was used to disrupt nearly all the GPCR and neuropeptide genes from C. elegans genome. Multiple genes were disrupted in each strain for the purpose of screening. The genotype is the list of targeted genes
Project description:G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and weaker phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with weaker phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.
Project description:The aim of this part of the wider project is to identify neuropeptide precursors, investigate cleavage sites on neuropeptide precursors and predict mature peptides in the sea anemone Nematostella vectensis. This was done to create a synthetic library of N. vectensis neuropeptides which were then used to test neuropeptide receptor candidates for activation by the different peptides.
Project description:Cells carrying lentiCRISPRv2 e.v. or lentiCRISPRv2 sgSTING were selected with puromycin before DNA was extracted to assess the genomic alterations at the target locus.
Project description:This study shows that chemically and pharmacodynamically distinct agonists acting on the same GPCR can produce indistinguishable cellular responses and that this uniformity is conferred by endosomal signaling The ability of chemically distinct ligands to produce different effects on the same G protein-coupled receptor (GPCR) has interesting therapeutic implications but, if excessively propagated downstream, would introduce biological 'noise' compromising cognate ligand detection We asked if cells have the ability to limit the degree to which chemical diversity imposed at the ligand-GPCR interface is propagated to the downstream signal We carried out an unbiased analysis of the integrated cellular response elicited by two chemically and pharmacodynamically diverse β-adrenoceptor agonists, isoproterenol and salmeterol We show that both ligands generate an identical integrated response, and that this stereotyped output requires endocytosis We further demonstrate that the endosomal β2-AR signal confers uniformity on the downstream response because it is highly sensitive and saturable Based on these findings, we propose that GPCR signaling from endosomes functions as a biological noise filter to enhance reliability of cognate ligand detection
Project description:In human HCC tumors, matching paracancerous specimens, and normal liver specimens, a targeted gene expression microanalysis was used to screen alterations in GPCR family gene expression.
Project description:T helper type 1 (Th1) cells play critical roles in viral infection response and autoimmune disease. Recent studies have implicated the imbalance of T helper subtypes is correlated with outcome of COVID-19 infection, however, the underlying mechanism that balancing Th1 cells versus other T helper cells remains unknown. Here, we profiled the heterogeneity and dynamic regulation during in vivo and in vitro Th1 differentiation using scRNA-seq and defined the Th1 unique program using a Th1/Th2 dichotomous differentiation condition. To further validate the functions of defined Th1 regulators, we performed CRISPR screenings for both in vitro as well as in vivo Th1 differentiation. RAMP3 as one of the receptors for neuropeptide CGRP was defined as a novel regulator balancing Th1 and Th2 differentiation and was proved an indispensable role for Th1 response during virus infection. CGRP, RAMP3 and cAMP signaling form a positive feedback loop to enhance Th1 differentiation through activating STAT1. Together, ur work highlights the network between CGRP-mediated neuronal signaling and immune response during virus infection.