小W
(2022-07-31 09:59):
#paper doi：https ://doi.org/10.1016/j.cell.2022.05.013
Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq
Perturb-seq 是一种实验方法，通过将基于 CRISPR 的遗传筛选与单细胞 RNA 测序表型相结合，绘制遗传扰动的转录效应。本文使用 CRISPRi 靶向慢性髓性白血病细胞（K562）中的所有表达基因和视网膜色素上皮细胞（RPE1）中的所有 DepMap（癌症依赖关系分析数据库） 必需基因，基于其CRISPRi基因-RNA表型的内在可解释性，将基因与它在细胞中的作用联系起来。阐述了Perturb-seq基因组筛选在以下方向的应用：1.预测引起转录表型的遗传扰动特征；2.从转录表型注释基因功能；3.复合表型假设驱动研究；4.线粒体基因组应激特异性调控。本文是使用Perturb-seq 技术对每个基因的遗传扰动分析，其测序数据以及表达（和差异分析）数据、sgRNA库(未找到)已公布，主要实验方法和分析脚本参照另一篇论文 "Scalable single-cell CRISPR screens by direct guide RNA capture and targeted library enrichment, Nature Biotechnology 2020"。
Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq
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Abstract:
A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.
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