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Series GSE129282 Query DataSets for GSE129282
Status Public on May 01, 2019
Title CRISPR knockout screen implicates three genes in lysosome function
Organisms Homo sapiens; synthetic construct
Experiment type Other
Summary 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.
Overall design We performed a CRSIPR screen to identify genes that, when mutated, result in accumulation of enlarged vacuoles.
Contributor(s) Kitzman JO
Citation(s) 31270356
Submission date Apr 03, 2019
Last update date Jul 31, 2019
Contact name Jacob Kitzman
Organization name University of Michigan
Department Department of Human Genetics
Lab Kitzman Lab
Street address 4811 Med Sci II, 1241 E Catherine St
City Ann Arbor
State/province MI
ZIP/Postal code 48109
Country USA
Platforms (3)
GPL15520 Illumina MiSeq (Homo sapiens)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
GPL19604 Illumina HiSeq 2500 (synthetic construct)
Samples (9)
GSM3703947 GECKOv2 plasmid pool A
GSM3703948 GECKOv2 plasmid pool B
GSM3703949 Transduced cell population
BioProject PRJNA530740
SRA SRP190447

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Supplementary file Size Download File type/resource
GSE129282_joined.counts.txt.gz 1.1 Mb (ftp)(http) TXT
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Raw data are available in SRA
Processed data are available on Series record

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