Caroline I. Fandrey, Marius Jentzsch, Peter Konopka, Alexander Hoch, Katja Blumenstock, Afraa Zackria, Salie Maasewerd, Marta Lovotti, Dorothee J. Lapp, Florian N. Gohr, Piotr Suwara, Jędrzej Świeżewski, Lukas Rossnagel, Fabienne Gobs, Maia Cristodaro, Lina Muhandes, Rayk Behrendt, Martin C. Lam, Klaus J. Walgenbach, Tobias Bald, Florian I. Schmidt, Eicke Latz, Jonathan L. Schmid-Burgk
{"title":"NIS-Seq enables cell-type-agnostic optical perturbation screening","authors":"Caroline I. Fandrey, Marius Jentzsch, Peter Konopka, Alexander Hoch, Katja Blumenstock, Afraa Zackria, Salie Maasewerd, Marta Lovotti, Dorothee J. Lapp, Florian N. Gohr, Piotr Suwara, Jędrzej Świeżewski, Lukas Rossnagel, Fabienne Gobs, Maia Cristodaro, Lina Muhandes, Rayk Behrendt, Martin C. Lam, Klaus J. Walgenbach, Tobias Bald, Florian I. Schmidt, Eicke Latz, Jonathan L. Schmid-Burgk","doi":"10.1038/s41587-024-02516-5","DOIUrl":null,"url":null,"abstract":"<p>Optical pooled screening offers a broader-scale alternative to enrichment-based perturbation screening, using fluorescence microscopy to correlate phenotypes and perturbations across single cells. Previous methods work well in large, transcriptionally active cell lines, because they rely on cytosolic detection of endogenously expressed barcoded transcripts; however, they are limited by reliable cell segmentation, cytosol size, transcriptional activity and cell density. Nuclear In-Situ Sequencing (NIS-Seq) expands this technology by creating bright sequencing signals directly from nuclear genomic DNA to screen nucleated cells at high density and high library complexity. By inserting an inverted phage promoter downstream of the single guide RNA (sgRNA), many RNA copies of the sgRNA can be generated and sequenced independently of cellular transcription. In this study, we benchmarked NIS-Seq across eight cell types from two species and performed four genome-scale optical perturbation screens, identifying key players of inflammation-related cellular pathways. Finally, we performed a small-scale pooled optical screen in primary human macrophages from blood of healthy donors and demonstrated barcode identification in lentivirally transduced human skin tissue.</p>","PeriodicalId":19084,"journal":{"name":"Nature biotechnology","volume":"40 1","pages":""},"PeriodicalIF":33.1000,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1038/s41587-024-02516-5","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Optical pooled screening offers a broader-scale alternative to enrichment-based perturbation screening, using fluorescence microscopy to correlate phenotypes and perturbations across single cells. Previous methods work well in large, transcriptionally active cell lines, because they rely on cytosolic detection of endogenously expressed barcoded transcripts; however, they are limited by reliable cell segmentation, cytosol size, transcriptional activity and cell density. Nuclear In-Situ Sequencing (NIS-Seq) expands this technology by creating bright sequencing signals directly from nuclear genomic DNA to screen nucleated cells at high density and high library complexity. By inserting an inverted phage promoter downstream of the single guide RNA (sgRNA), many RNA copies of the sgRNA can be generated and sequenced independently of cellular transcription. In this study, we benchmarked NIS-Seq across eight cell types from two species and performed four genome-scale optical perturbation screens, identifying key players of inflammation-related cellular pathways. Finally, we performed a small-scale pooled optical screen in primary human macrophages from blood of healthy donors and demonstrated barcode identification in lentivirally transduced human skin tissue.
期刊介绍:
Nature Biotechnology is a monthly journal that focuses on the science and business of biotechnology. It covers a wide range of topics including technology/methodology advancements in the biological, biomedical, agricultural, and environmental sciences. The journal also explores the commercial, political, ethical, legal, and societal aspects of this research.
The journal serves researchers by providing peer-reviewed research papers in the field of biotechnology. It also serves the business community by delivering news about research developments. This approach ensures that both the scientific and business communities are well-informed and able to stay up-to-date on the latest advancements and opportunities in the field.
Some key areas of interest in which the journal actively seeks research papers include molecular engineering of nucleic acids and proteins, molecular therapy, large-scale biology, computational biology, regenerative medicine, imaging technology, analytical biotechnology, applied immunology, food and agricultural biotechnology, and environmental biotechnology.
In summary, Nature Biotechnology is a comprehensive journal that covers both the scientific and business aspects of biotechnology. It strives to provide researchers with valuable research papers and news while also delivering important scientific advancements to the business community.