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新方法可用于剖析遗传网络
作者:小柯机器人 发布时间:2020/12/12 20:52:17

美国加州大学伯克利分校Nicholas T. Ingolia研究小组开发出CiBER-seq技术,可用于解剖遗传网络。这一研究成果于2020年12月11日发表在国际学术期刊《科学》上。

据研究人员介绍,为了使用基于CRISPR-Cas9的遗传学,人们需要采取方法来量化整个基因组全基因扰动文库中特定的分子表型。

通过使用带有条形码表达报告基因测序的CRISPR干扰(CiBER-seq)技术,研究人员分析了转录、翻译和翻译后报告基因来解决这一难题。这个条形码方法使得研究人员可以使用合并测序将整个向导库与各自的表型后果联系起来。CiBER-seq分析完全概括了酵母中的整合应激反应(ISR)途径。遗传扰动导致无负载的转移RNA(tRNA)积累,从而激活了ISR报告基因转录。

值得注意的是,tRNA功能不全也会激活报告基因,而与无负载的tRNA感应蛋白无关。通过揭示ISR激活的选择性触发因素,研究人员证明了精确、全面的CiBER-seq分析如何为解剖遗传网络提供了一个强大而广泛适用的工具。

附:英文原文

Title: CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

Author: Ryan Muller, Zuriah A. Meacham, Lucas Ferguson, Nicholas T. Ingolia

Issue&Volume: 2020/12/11

Abstract: To realize the promise of CRISPR-Cas9–based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

DOI: 10.1126/science.abb9662

Source: https://science.sciencemag.org/content/370/6522/eabb9662

期刊信息
Science:《科学》,创刊于1880年。隶属于美国科学促进会,最新IF:41.037