2. Academic Validation
  3. NIS-Seq enables cell-type-agnostic optical perturbation screening

NIS-Seq enables cell-type-agnostic optical perturbation screening

  • Nat Biotechnol. 2024 Dec 19. doi: 10.1038/s41587-024-02516-5.
Caroline I Fandrey # 1,  Marius Jentzsch # 1,  Peter Konopka 1,  Alexander Hoch 1,  Katja Blumenstock 1,  Afraa Zackria 1,  Salie Maasewerd 2,  Marta Lovotti 2,  Dorothee J Lapp 2 3,  Florian N Gohr 2 3,  Piotr Suwara 4,  Jędrzej Świeżewski 4,  Lukas Rossnagel 2,  Fabienne Gobs 1,  Maia Cristodaro 1,  Lina Muhandes 1,  Rayk Behrendt 1,  Martin C Lam 5,  Klaus J Walgenbach 5,  Tobias Bald 6,  Florian I Schmidt 2,  Eicke Latz 2 7,  Jonathan L Schmid-Burgk 8
Affiliations

Affiliations

  • 1 Institute of Clinical Chemistry and Clinical Pharmacology, University and University Hospital Bonn, Bonn, Germany.
  • 2 Institute of Innate Immunity, University and University Hospital Bonn, Bonn, Germany.
  • 3 Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.
  • 4 Appsilon, Warszawa, Poland.
  • 5 Division of Plastic, Reconstructive and Aesthetic Surgery, Department of Surgery, University and University Hospital Bonn, Bonn, Germany.
  • 6 Institute of Experimental Oncology, University and University Hospital Bonn, Bonn, Germany.
  • 7 German Leibniz Centre for Rheumatism Research, Berlin, Germany.
  • 8 Institute of Clinical Chemistry and Clinical Pharmacology, University and University Hospital Bonn, Bonn, Germany. jsb@uni-bonn.de.
  • # Contributed equally.
PMID: 39702735 DOI: 10.1038/s41587-024-02516-5
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.

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