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High-precision pooled screening for quantitative molecular phenotypes

定量分子表型的高精度联合筛选

基本信息

批准号：
10245276
负责人：
NICHOLAS T INGOLIA
金额：
$ 30.71万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10245276
关键词：
Address Affect Bar Codes Biological Biological Process CRISPR screen CRISPR/Cas technology Cell physiology Cells Chromosome Mapping Complex DNA Data Dependence Engineering Event Gene Expression Gene Silencing Genes Genetic Genetic Epistasis Genetic Screening Genetic Transcription Genome Growth Guide RNA Human Individual Label Lead Learning Libraries Link Mammalian Cell Measurement Measures Molecular Monitor Mutagenesis Mutation Nucleotides Organism Outcome Pathway interactions Pattern Phenotype Population Population Sizes Process Proteins RNA Reagent Regulation Reporter Reporting Resolution Resources Saccharomycetales Signal Pathway Signal Transduction Sorting - Cell Movement Source Specific qualifier value System Techniques Transgenic Organisms Translating Translations cell growth computerized tools deep sequencing experimental study gene function genetic profiling genome-wide insight interest molecular phenotype novel precision genetics programs promoter response screening tool transcription factor

项目摘要

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.

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，提供了广泛有用的资源，高精度遗传 profiling.