High-precision pooled screening for quantitative molecular phenotypes

摘要 CRISPR/Cas9的RNA引导的DNA识别现在可以实现全面的遗传 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提供了 通过DNA切割、转录抑制或 定向诱变测量与这些遗传扰动相关的表型 仍然是一个挑战，这通常是绘制基因图谱的最大障碍。 重要生物过程的依赖性。 我们建议开发一种通用方法，将分子表型与靶向 在大的合并细胞群中诱导这些效应的指导RNA。我们将不同的指南 具有独特核苷酸条形码的RNA，其在RNA报告基因中表达，将RNA 在一些实施方案中，所述条形码的丰度与感兴趣的细胞内过程相关。我们的系统可以直接监控 转录，转录后和翻译后反应，使我们能够耦合它， 细胞内信号的广泛排列这些表达测量非常适合于 上位性分析，可以识别遗传途径并发现新的基因功能 通过数量表型特征的遗传相互作用的相关模式。我们将 提供我们经过验证的试剂，用于在萌芽中实施这种筛选方法 酵母和哺乳动物细胞中，为高精度遗传学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.