白鸟
(2022-11-30 11:26):
#paper https://doi.org/10.1016/j.cell.2022.05.013 Cell 2022. Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. 遗传学的一个核心目标是研究遗传变化(基因型)和表型之间的关系。主要有两种研究思路，正向遗传学和反向遗传学。正向遗传以表型为中心的“正向遗传”，即揭示驱动表型的基因变化（果因论）；而反向遗传是以基因为中心，对确定的遗传变化引起的不同表型进行解析（因果论）。为了揭示基因扰动的功能后果和基因型-表型关系，文章团队构建了一套可实践的方法论。本文利用单细胞高通量CRISPR 筛选技术Perturb-seq，针对对K562和RPE1细胞系超过250万个细胞进行了单个基因的CRISPR扰动（即1个细胞只包含一种基因的 sgRNA），通过单一基因型的变化，查看在转录组层面表型的变化，构建了一个基因型-表型综合图谱。研究团队根据基因的共同调控将其聚类到特定表达程序中，并计算每个扰动簇中每个基因表达程序的平均活性。分析结果包含多个与基因干扰相关的已知表达程序，包括蛋白酶体功能障碍导致的蛋白酶体亚基上调、 ESCRT蛋白缺失时NF-kB信号通路的激活，以及胆固醇生物合成上调对囊泡运输缺陷的反应等。它的意义在于单细胞CRISPR筛选为系统探索遗传和细胞功能提供了一个研究工具，构建和分析丰富的基因型-表现型图谱,以作为系统探索遗传和细胞功能的驱动力。可以构建全基因组的基因敲除细胞池，定向的研究，关键基因的敲除对下游转录调控表型的生物学功能。重点学习文章中grna的数据质控和归一化等细节处理。
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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