期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：22
页码：10842-10851
DOI：10.1073/pnas.1903808116
出版社：The National Academy of Sciences of the United States of America
摘要：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.
