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.

光学混合筛选为基于富集的扰动筛选提供了更大规模的替代方案，使用荧光显微镜来关联单个细胞的表型和扰动。以前的方法在转录活性的大细胞系中效果很好，因为它们依赖于内源性表达的条形码转录本的胞质检测;然而，它们受到可靠的细胞分割、胞质溶胶大小、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 创建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。通过在单向导 RNA （sgRNA） 的下游插入倒置噬菌体启动子，可以独立于细胞转录生成和测序 sgRNA 的许多 RNA 拷贝。在这项研究中，我们对来自两个物种的 8 种细胞类型的 NIS-Seq 进行了基准测试，并进行了 4 次基因组规模的光学扰动筛选，确定了炎症相关细胞通路的关键参与者。最后，我们在来自健康供体血液的原代人巨噬细胞中进行了小规模的混合光学筛选，并证明了慢病毒转导的人皮肤组织中的条形码识别。