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新技术能够在单细胞中实现基因组规模的遗传筛选
作者:小柯机器人 发布时间:2020/6/2 20:37:27

2020年6月1日,德国欧洲分子生物学实验室(EMBL)Lars M. Steinmetz、西班牙巴塞罗那科技学院Lars Velten等研究人员合作在《自然—方法学》杂志在线发表论文,报道了一种能够在单细胞中实现基因组规模遗传筛选的技术。

研究人员表示,转录组包含有关分子、细胞和生物表型的丰富信息。然而,实验和统计的限制约束了单细胞转录组学遗传筛选的敏感性和通量。

为了克服这些限制,研究人员开发了一个名为targeted Perturb-seq(TAP-seq)的方法,这是一种敏感、便宜且不依赖平台的方法,可将单细胞RNA-seq覆盖集中在目标基因上,从而按顺序提高了基因筛选的敏感性和规模数量级。

TAP-seq能够在单个实验中对成千上万个CRISPR介导的扰动进行常规分析,检测弱效应和低表达基因,并将测序要求降低多达50倍。研究人员应用TAP-seq为人类基因组2.5%内的1,778个增强子生成了基于扰动的增强子-靶基因图谱。

因此,研究人员表明,增强子与靶标的结合是由三维接触频率和表观遗传状态共同决定的,从而可以准确预测整个基因组中的增强子靶标。此外,研究人员证明TAP-seq可以鉴别每个细胞中仅100个测序读数的细胞亚型。

附:英文原文

Title: Targeted Perturb-seq enables genome-scale genetic screens in single cells

Author: Daniel Schraivogel, Andreas R. Gschwind, Jennifer H. Milbank, Daniel R. Leonce, Petra Jakob, Lukas Mathur, Jan O. Korbel, Christoph A. Merten, Lars Velten, Lars M. Steinmetz

Issue&Volume: 2020-06-01

Abstract: The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer–target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer–target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.

DOI: 10.1038/s41592-020-0837-5

Source: https://www.nature.com/articles/s41592-020-0837-5

期刊信息

Nature Methods:《自然—方法学》,创刊于2004年。隶属于施普林格·自然出版集团,最新IF:28.467
官方网址:https://www.nature.com/nmeth/
投稿链接:https://mts-nmeth.nature.com/cgi-bin/main.plex