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| Status |
Public on May 01, 2019 |
| Title |
CRISPR knockout screen implicates three genes in lysosome function |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| 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.
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| Overall design |
We performed a CRSIPR screen to identify genes that, when mutated, result in accumulation of enlarged vacuoles.
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| Contributor(s) |
Kitzman JO |
| Citation(s) |
31270356 |
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| Submission date |
Apr 03, 2019 |
| Last update date |
Jul 31, 2019 |
| Contact name |
Jacob Kitzman |
| Organization name |
University of Michigan
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| Department |
Department of Human Genetics
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| Lab |
Kitzman Lab
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| Street address |
4811 Med Sci II, 1241 E Catherine St
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platforms (3) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA530740 |
| SRA |
SRP190447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE129282_joined.counts.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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