Defining gene-by-environment interactions using multiplex single-cell genomics

项目摘要 拟议的研究计划旨在开发系统的遗传效应的多重评估， 干扰细胞对外源性和内源性暴露的反应， 细胞 .具体来说，我们将开发一个灵活的和可扩展的体外平台，用于高通量单细胞基因， 通过环境相互作用筛选，将基于CRISPR的单细胞合并遗传筛选与“核基因筛选”相结合。 基于“散列”的样品多重化策略和单细胞基因组读出。该平台将允许 同时对1,000 - 10,000个独特条件（即基因和暴露）进行分子描述 组合）在单个细胞类型和细胞状态的分辨率下。此外，我们建议制定一套 基于异源病毒受体表达的工具，用于CRISPR的原位和细胞类型特异性递送- 以与单细胞RNA-seq兼容的方式基于遗传扰动。我们强调多功能性， 我们的方法在我们的技术的三个应用中的可推广性，这些技术旨在通过 神经生物学疾病中的环境相互作用。我们建议剖析基因之间的相互作用， 与阿尔茨海默病相关的突变和神经细胞类型对氧化应激的转录反应 应力和肽聚集体;表征与细胞类型特异性 响应于引起癫痫病症的基因中的突变的遗传修饰剂活性的调节;以及 确定脑癌中常见的突变基因如何改变小鼠肿瘤对 体内治疗我们的目标是达到临床上可操作的分子描述， 有助于个体对暴露的反应。

PROJECT SUMMARY The proposed research program aims to develop systems for the multiplex assessment of the effect of genetic perturbation on the response of cells to exogenous and endogenous exposures at the resolution of individual cells . Specifically, we will develop a flexible and scalable in vitro platform for high-throughput single-cell gene- by-environment interaction screens that couples CRISPR-based single-cell pooled genetic screens to a “nuclear hashing”-based sample multiplexing strategy and a single-cell genomic readout. This platform will allow for the simultaneous molecular description of 1,000s-10,000s of unique conditions (i.e. gene and exposure combinations) at the resolution of individual cell types and cell states. In addition, we propose to develop a set of tools based on heterologous viral receptor expression for the in situ and cell-type specific delivery of CRISPR- based genetic perturbations in a manner compatible with single-cell RNA-seq. We highlight the versatility and generalizability of our approach across three applications of our technologies that aim to define gene-by- environment interactions in neurobiological disease. We propose to dissect the genetic interactions between mutations associated with Alzheimer’s disease and the transcriptional response of neural cell types to oxidative stress and peptide aggregates; to characterize the transcriptional effects associated with cell-type specific modulation of genetic modifier activity in response to mutations in genes that cause epilepsy disorder and; to determine how genes that are commonly mutated in brain cancer alter the response of murine tumors to therapies in vivo. Our goal is to arrive at clinically actionable molecular descriptions of how individual genes contribute to the response of individuals to exposure.