Perturb-FISH simultaneous CRISPR screening and spatial transcriptomics in astrocytes

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

汇集式光学筛选已实现大规模细胞互作、形态及动态特征的研究，但尚未借助高多重化单细胞分辨率转录组读数的能力来解析分子通路。本研究报道了Perturb-FISH技术，其通过将成像空间转录组学与原位扩增向导RNA（guide RNA）的并行光学检测相结合，打通了两类研究路径的衔接壁垒。研究显示，在针对培养单核细胞的脂多糖（lipopolysaccharide）应答筛选实验中，Perturb-FISH所获取的胞内效应与Perturb-seq结果高度吻合，同时还揭示了先天免疫应答中全新的细胞间及密度依赖性调控机制。本研究进一步将Perturb-FISH与功能检测读数相结合，开展自闭症谱系障碍（autism spectrum disorder）风险基因筛选实验，在诱导多能干细胞（induced pluripotent stem cell）衍生的星形胶质细胞中发现了共通的钙活性表型，以及与之相关的遗传互作与失调分子通路。综上，Perturb-FISH是一种通用的研究方法，可在单细胞分辨率下解析空间生物学与功能生物学的遗传及分子关联。

搜集汇总

背景与挑战

背景概述

该数据集介绍了Perturb-FISH方法，该方法将成像空间转录组学与CRISPR筛选结合，实现单细胞分辨率下的空间转录组学和功能研究。它已应用于研究免疫反应和自闭症风险基因，揭示遗传相互作用和分子通路。