Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E. Mohr, Norbert Perrimon
{"title":"果蝇细胞中的CRISPR筛选","authors":"Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E. Mohr, Norbert Perrimon","doi":"10.1002/cpmb.111","DOIUrl":null,"url":null,"abstract":"<p>High-throughput screens in <i>Drosophila melanogaster</i> cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in <i>Drosophila</i> S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in <i>Drosophila</i>, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc.</p><p><b>Basic Protocol</b>: Pooled-format screening with Cas9-expressing <i>Drosophila</i> S2R+ cells in the presence of cytotoxin</p><p><b>Support Protocol 1</b>: Optimization of cytotoxin concentration for <i>Drosophila</i> cell screening</p><p><b>Support Protocol 2</b>: CRISPR sgRNA library design and production for <i>Drosophila</i> cell screening</p><p><b>Support Protocol 3</b>: Barcode deconvolution and analysis of screening data</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"129 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2019-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.111","citationCount":"12","resultStr":"{\"title\":\"Pooled CRISPR Screens in Drosophila Cells\",\"authors\":\"Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E. Mohr, Norbert Perrimon\",\"doi\":\"10.1002/cpmb.111\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>High-throughput screens in <i>Drosophila melanogaster</i> cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in <i>Drosophila</i> S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in <i>Drosophila</i>, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc.</p><p><b>Basic Protocol</b>: Pooled-format screening with Cas9-expressing <i>Drosophila</i> S2R+ cells in the presence of cytotoxin</p><p><b>Support Protocol 1</b>: Optimization of cytotoxin concentration for <i>Drosophila</i> cell screening</p><p><b>Support Protocol 2</b>: CRISPR sgRNA library design and production for <i>Drosophila</i> cell screening</p><p><b>Support Protocol 3</b>: Barcode deconvolution and analysis of screening data</p>\",\"PeriodicalId\":10734,\"journal\":{\"name\":\"Current Protocols in Molecular Biology\",\"volume\":\"129 1\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-11-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpmb.111\",\"citationCount\":\"12\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current Protocols in Molecular Biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpmb.111\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Molecular Biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpmb.111","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 12
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