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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：
10058222
负责人：
NICHOLAS T INGOLIA
金额：
$ 30.53万
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/10058222
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 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。