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

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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Publication Details

Journal
Nature Biotechnology
Published
2024-12-19
DOI
https://doi.org/10.1038/s41587-024-02516-5
Citations
14
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
3.27

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article

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

Marius Jentzsch, Eicke Latz, Afraa Aqeel Zackria, Marta Lovotti et al.
14 citations
Nature Biotechnology
CRISPR and Genetic Engineering
3.27
article

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

Marius Jentzsch, Eicke Latz, Afraa Aqeel Zackria, Marta Lovotti, Lina Muhandes, Tobias Bald, Rayk Behrendt, Jonathan L. Schmid‐Burgk, Caroline I. Fandrey, Florian Ingo Schmidt, Katja Blumenstock, Piotr Suwara, Florian N. Gohr, Alexander Hoch, Salie Maasewerd, Dorothee J. Lapp, Martin C. Lam, Jędrzej Świeżewski, Lukas Roßnagel, Peter Konopka, Fabienne Gobs, Maia Cristodaro, Klaus J. Walgenbach
article en
14 citations

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.

Nature BiotechnologyVol. 43(8)
The University of Melbourne (AU), University Hospital Bonn (DE), German Rheumatism Research Centre (DE), Peter Doherty Institute (AU)
Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, Rheinische Friedrich-Wilhelms-Universität Bonn, Leibniz-Gemeinschaft, Institute of Infection and Immunity
Openalex Percentile: Top 7%
CRISPR and Genetic Engineering
3.27
DOIOpen Access PDFOpenAlex
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