CRISPR knockout screen implicates three genes in lysosome function

Abstract

Defective biosynthesis of the phospholipid PI(3,5)P 2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived 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. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P 2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35 , LRRC8A , and MARCH7 . We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.

Endomembrane Tension and Trafficking

“…Interestingly, gain of function mutations in members of the ClC family result in volume increases in the endocytic pathway (Nicoli et al, 2019 ), and ClCs are generally outwardly rectifying, so while these transporters have yet to show a role in the rapid process of volume resolution, they may contribute to the osmoregulation of endolysosomes in other ways. In addition, a recent screen identified LRRC8A, a component of the volume-regulated anion channel (VRAC), as necessary for the control of vacuolar volume, since inactivation of the LRRC8A gene causes swelling of endolysosomes (Lenk et al, 2019 ). Whether this effect is directly caused by the loss of VRAC activity from endosomes remains to be formally tested.…”

Improving gene function predictions using independent transcriptional components

“…In addition, we investigated three genome-wide CRISPR screens that aimed to capture genes involved in the inflammatory response to bacterial lipopolysaccharide (LPS) 19 , lysosomal accumulation 20 , and viral entry 21 .…”

“…In the genome-wide CRISPR-knockout screen identifying 16 genes essential for lysosomal integrity, a network was built with GO Biological Process and KEGG gene set collections 20 . The ICA-TC-based prediction scores of the 16 genes for KEGG lysosome gene set ranged from 0.8 to 2.6, indicating that the ICA-TC method did not confidently predict these genes to play a role in lysosome-related processes (Fig.…”

Essential genes from genome-wide screenings as a resource for neuropsychiatric disorders gene discovery

“…Because of these advantages, there have been many studies employing cancer cell line screenings to investigate biological processes other than cancer. For example, Lenk et al used genome-wide screening of essential genes in cancer cell lines to study lysosome function 42 , while Patel et al used a genome-wide screening approach to study antigen presentation and interferon-γ signaling 11 , both of which are pathways translatable to non-cancer cells.…”