哺乳动物细胞遗传相互作用网络的高通量构建方法

A genetic interaction refers to a functional relationship between individual genes, which can be determined by comparing phenotypes of individual genetic mutants with the phenotype of a combined genetic mutant. Systematic identification of genetic interactions in mammalian cells will not only help to improve our understanding of mammalian biological phenomena and human diseases, but also promote the development of novel therapeutic strategies. However, systematic screens of genetic interactions in mammalian cells are still limited by lack of efficient and high-throughput methods for constructing libraries of genetic pools that perturb biological systems in a defined manner, as well as consequent phenotype recording. Furthermore, no systematic approach has been developed to screen genetic interactions between multiple genes. In this project, we propose to develop an efficient and high-throughput method to construct libraries of double-mutant pools by using CRISPR/Cas system. In addition, we will investigate whether synthetic microRNA cluster can be used to simultaneously silence multiple gene expression and develop a method to efficiently assemble libraries of synthetic microRNA clusters for systematic screens of genetic interactions between functionally redundant genes. We will also develop novel computational methods to analyze genetic interaction networks by using phenotypic data obtained via newly developed double-knockout or multi-knockdown techniques. This project will provide novel strategies for systematic screens of mammalian genetic interactions in a biological network with or without functional redundancies.

遗传相互作用是指基因间的功能关系，可以通过研究单个基因沉默后的表型与组合基因沉默后的表型差异来确定。研究哺乳动物细胞中分子网络节点间的遗传相互作用，将对阐述哺乳动物细胞的生命现象，发现疾病发病机制和寻找治疗方案等提供重要依据。然而，采用高通量的基因表达沉默技术，在哺乳动物细胞中研究双基因遗传相互作用的方法还处在探索初期，并且目前还没有研究多基因遗传相互作用的高通量方法。本项目将针对这一国际前沿问题，建立基于CRISPR/Cas技术的双基因敲除文库构建和表型观测高通量实验方法；研究利用合成microRNA簇敲低多个基因的可行性，建立microRNA簇文库的快速组装和多基因敲低的表型观测方法；探索基于双基因敲除和多基因敲低技术构建遗传相互作用网络的计算分析方法。本项目的顺利进行，将大大提高哺乳动物细胞遗传相互作用网络研究的效率，并将为功能冗余基因间相互作用的研究提供高效实验和分析方法。

研究哺乳动物细胞中分子网络节点间的功能相互作用，将对阐述哺乳动物细胞的生命现象，发现疾病发病机制和寻找治疗方案等提供重要依据。本课题建立了CRISPR/Cas双gRNA文库的构建方法和功能筛选方法，研究了黑色素瘤对威罗菲尼耐药性和与结直肠炎症向肿瘤发生过程有关的遗传相互作用网络。我们还建立了microRNA簇的快速组装合成方法和细胞功能筛选方法。此外，我们还建立了一套高通量双敲除和多敲低数据的生物信息学分析流程.本课题的研究方法将为后续哺乳动物细胞的遗传相互作用网络研究提供了高效工具和参考。