Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization
| Autor: | George Emanuel, Tian Lu, Chong Wang, Xiaowei Zhuang, Hazen P. Babcock |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Untranslated region
RNA localization Computational biology Biology high-throughput screening 03 medical and health sciences 0302 clinical medicine Cell Line Tumor Image Processing Computer-Assisted CRISPR Humans Guide RNA MERFISH nuclear compartments In Situ Hybridization Fluorescence 030304 developmental biology Subgenomic mRNA Cell Nucleus Gene Editing 0303 health sciences Reporter gene MALAT1 Multidisciplinary High-Throughput Nucleotide Sequencing RNA-Binding Proteins Cell Biology Biological Sciences Long non-coding RNA 3. Good health PNAS Plus Molecular Probes RNA Long Noncoding CRISPR-Cas Systems Single-Cell Analysis 030217 neurology & neurosurgery RNA Guide Kinetoplastida |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 |
| Popis: | Significance In this work, we developed an imaging-based pooled-library CRISPR screening approach that provides readouts of both phenotype and genotype of individual cells by high-resolution, high-content imaging. This approach promises to substantially expand the phenotype space accessible to pooled genetic screening by allowing the probing of complex cellular phenotypes, such as cell morphology and subcellular organization of different molecular species, as well as their dynamics. Applying this approach to screen for genetic factors involved in nuclear RNA localization, we identified both positive and negative regulators that control lncRNA localization to nuclear speckles. Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3′ untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments. |
| Databáze: | OpenAIRE |
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