Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Science. 2020 Dec 11;370(6522). doi: 10.1126/science.abb9662.
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.
为了实现基于 CRISPR-Cas9 的遗传学的承诺，需要采用方法来量化全基因组遗传干扰文库中特定的分子表型。我们通过使用带有条形码表达报告基因测序（CiBER-seq）的 CRISPR 干扰（CRISPRi）来对转录、翻译和翻译后报告基因进行分析，从而解决了这一挑战。我们的条形码方法允许我们使用池测序将整个向导文库与其单个表型后果联系起来。CiBER-seq 分析完全再现了酵母中的综合应激反应（ISR）途径。导致无电荷转移 RNA（tRNA）积累的遗传干扰激活了 ISR 报告基因的转录。值得注意的是，无电荷 tRNA 传感器独立于 tRNA 不足也会激活报告基因。通过揭示 ISR 激活的替代触发因素，我们说明了精确、全面的 CiBER-seq 分析如何为剖析遗传网络提供强大且广泛适用的工具。
