High-precision pooled screening for quantitative molecular phenotypes

摘要 RNA引导的CRISPR/Cas9的DNA识别现在使全面的基因 在哺乳动物细胞和其他以前难以处理的系统中进行筛选。CRISPR/CAS9提供 高度可编程的通过DNA切割、转录抑制或 有针对性的突变。测量与这些遗传扰动相关的表型 仍然是一个挑战，这往往是绘制基因图谱的最大障碍 重要生物过程的依赖性。 我们建议开发一种将分子表型与靶向联系起来的一般方法。 引导在大量汇集的细胞群体中产生这些影响的RNA。我们把不同的指南贴上标签 具有独特核苷酸条形码的RNA，在RNA报告程序中表达，连接RNA 丰富的条形码连接到感兴趣的细胞内过程。我们的系统可以直接监控 转录、转录后和翻译后反应，使我们能够将其结合在一起 具有广泛的细胞内信号。这些表情测量结果非常适合于 上位性分析，可以识别遗传途径并发现新的基因功能 通过来自数量表型的遗传交互作用的相关模式，fiLes。我们会 提供我们经过验证的试剂，用于在萌芽阶段实施这种筛选方法 酵母和哺乳动物细胞中，为高精度遗传提供了广泛有用的资源 ProfiLing。

ABSTRACT The RNA-guided DNA recognition of CRISPR/Cas9 now enables comprehensive genetic screening in mammalian cells and other previously intractable systems. CRISPR/Cas9 provides highly programmable genetic perturbation through DNA cleavage, transcriptional inhibition, or targeted mutagenesis. Measuring the phenotypes associated with these genetic perturbations remains a challenge, and this often represents the greatest barrier to mapping the genetic dependencies of important biological processes. We propose to develop a general approach that links molecular phenotypes with the targeting guide RNAs that induce those effects in a large, pooled cell population. We label distinct guide RNAs with unique nucleotide barcodes that are expressed in an RNA reporter, linking the RNA abundance of the barcode to intracellular processes of interest. Our system can directly monitor transcriptional, post-transcriptional, and post-translational responses, allowing us to couple it with a wide array of intracellular signals. These expression measurements are well suited for epistasis analysis, which can identify genetic pathways and uncover novel gene functions through correlated patterns of genetic interaction from quantitative phenotypic profiles. We will make available our validated reagents for implementing this screening approach in budding yeast and in mammalian cells, providing a broadly useful resource for high-precision genetic profiling.