Project description:Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large acidic vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. After calibration on cells lacking FIG4, a known PI(3,5)P2 biosynthetic factor, we carried out a genome-wide knockout screen that captured fifteen genes, including VAC14 which is known to cause endosomal vacuolization. We selected three genes not previously associated with lysosome dysfunction for validation: C10orf35, LRRC8A, and MARCH7. We isolated two clonal knockout cell lines for each gene and confirmed loss of protein expression. The knockout lines contained enlarged acidic vesicles with surface labeling of the endolysosomal marker LAMP2. This assay will be useful for characterizing variants of unknown significance in patients with neuromuscular or lysosomal storage disorders. This genome-wide strategy for identification of genes with lysosomal function can also be adapted for drug screens to identify small molecules that correct vacuolization.
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:To search for factors regulating neuronal differentiation, we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid human ESCs. The regulators were identified by the quantification of depletion of their mutant clones within a pooled loss-of-function library upon neuronal differentiation.
Project description:This is an in vitro genome-wide CRISPR/cas9 screen in human glioblastoma stem cells, screening for genes essential for survival of these cells. These cells express cas9 and have been transfected with a guide RNA library causing gene knockouts. We will analyse the sequencing data for depletion of guide RNAs.
Project description:We performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages to identify regulators of efferocytosis, the phagocytic clearance of dying cells. The screen identified known and novel regulators of macrophage efferocytosis. More broadly, the screen approach can be applied to interrogate complex functional phenotypes in primary macrophages.