德国波恩大学Jonathan L. Schmid-Burgk团队近期取得重要工作进展，他们研究开发了NIS-Seq技术，能实现与细胞类型无关的光学扰动筛选。相关研究成果2024年12月19日在线发表于《自然—生物技术》杂志上。

据介绍，光学混合筛选为基于富集的扰动筛选提供了一种更广泛的替代方案，使用荧光显微镜来关联单细胞的表型和扰动。以前的方法在较大的且转录活跃细胞系中效果良好，因为它们依赖于内源性表达的条形码转录物的细胞质检测；然而，它们受到可靠的细胞分割、细胞质大小、转录活性和细胞密度的限制。

核原位测序（NIS-Seq）通过直接从核基因组DNA中产生明亮的测序信号来扩展这项技术，以高密度和高文库复杂性筛选有核细胞。通过在单导向RNA（sgRNA）下游插入一个反向噬菌体启动子，可以独立于细胞转录产生和测序sgRNA的许多RNA拷贝。

研究人员对来自两个物种的八种细胞类型的NIS-Seq进行了基准测试，并进行了四次基因组规模的光学扰动筛选，确定了炎症相关细胞途径的关键参与者。最后，研究人员在健康供体血液中的原代人类巨噬细胞中进行了小规模的混合光学筛查，并在慢病毒转导的人类皮肤组织中进行了条形码识别。

附：英文原文

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

Author: Fandrey, Caroline I., Jentzsch, Marius, Konopka, Peter, Hoch, Alexander, Blumenstock, Katja, Zackria, Afraa, Maasewerd, Salie, Lovotti, Marta, Lapp, Dorothee J., Gohr, Florian N., Suwara, Piotr, wieewski, Jdrzej, Rossnagel, Lukas, Gobs, Fabienne, Cristodaro, Maia, Muhandes, Lina, Behrendt, Rayk, Lam, Martin C., Walgenbach, Klaus J., Bald, Tobias, Schmidt, Florian I., Latz, Eicke, Schmid-Burgk, Jonathan L.

Issue&Volume: 2024-12-19

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.

DOI: 10.1038/s41587-024-02516-5

Source: https://www.nature.com/articles/s41587-024-02516-5