Methods for mammalian genetic analysis

背景癌症研究需要我们能够准确地模拟在人类肿瘤中观察到的基因突变和基因表达模式。基因表达可以用RNAi下调。最近，CRISPR/Cas9已经成为一种主要的基因组编辑工具，用于在哺乳动物细胞中引入突变和改变基因表达。癌症基因组建模的一个主要挑战是，我们目前只能概括肿瘤细胞中观察到的复杂变化的一小部分。因此，开发能够在更复杂的规模上进行基因组编辑的方法对于临床前研究中的癌细胞行为建模至关重要。目标. 1.）开发用于同时下调人和小鼠细胞中多个基因表达的组合RNAi方法和试剂; 2）开发用于同时删除人和小鼠细胞中多个基因的组合CRISPR/Cas9基因敲除方法和试剂;以及3）开发用于在人和小鼠细胞中进行基因编辑以引入突变并调节基因表达的方法和试剂。主要活动、重大成果和关键成果。1）。用于组合基因敲低的多重RNAi。我们已经开发了使用高效siRNA的多重基因敲低方法。该方法能够在同一细胞中同时敲低多达7个基因。这种复杂程度使我们能够询问基因冗余和人类癌细胞中多个遗传节点的功能相互作用。这项工作已经出版。2）。用于组合基因敲除的多重CRISPR/Cas9。我们已经开发了使用预先验证的合成指导RNA的多重CIRSPR/Cas9基因敲除方法。这种方法能够同时敲低人类癌细胞中多达3个基因。这种方法能够在多个基因位点进行快速基因组编辑。这项工作已经出版。3）。开发用于遗传筛选的模块化CRISPR文库。我们已经开发了汇集的sgRNA文库模块的精选集合。每个模块中的基因通过其功能注释来管理。这使得能够构建亚基因组规模的、具有灵活大小的聚焦CRISPR文库，用于专门的遗传筛选目的。

BACKGROUND. Cancer research requires our ability to accurately model the pattern of gene mutation and gene expression observed in human tumors. Gene expression can be downregulated with RNAi. More recently, CRISPR/Cas9 has become a major genome-editing tool for introducing mutations and for altering gene expression in mammalian cells. A major challenge in modeling the cancer genome is that we can currently recapitulate only a fraction of the complex changes observed in a tumor cell. Thus, developing methods that enable genome editing on a more complex scale will be critical for the modeling of cancer cell behavior in pre-clinical studies. OBJECTIVES. 1.) To develop combinatorial RNAi method and reagents for downregulating the expression of multiple genes simultaneously in human and mouse cells; 2) To develop combinatorial CRISPR/Cas9 gene knockout method and reagent for deleting multiple genes simultaneously in human and mouse cells; and 3) to develop method and reagent for gene editing in human and mouse cells to introduce mutations and to regulate gene expression. MAJOR ACTIVITIES, SIGNIFICANT RESULTS AND KEY OUTCOMES. 1). Multiplexed RNAi for combinatorial gene knockdown. We have developed a multiplexed gene knockdown method using high potency siRNAs. This method enables the simultaneous knockdown of up to 7 genes in the same cell. This level of complexity allows us to interrogate gene paralog redundancy and the function interaction of multiple genetic nodes in human cancer cells. This work was published. 2). Multiplexed CRISPR/Cas9 for combinatorial gene knockout. We have developed a multiplexed CIRSPR/Cas9 gene knockout method using pre-validated, synthetic guide RNAs. This method enables the simultaneous knockdown of up to 3 genes in human cancer cells. This approach enables rapid genome editing at multiple gene loci. This work was published. 3). Development of modular CRISPR libraries for genetic screens. We have developed a curated collection of pooled sgRNAs library modules. Genes in each module were curated by their functional annotation. This enables the construction of sub-genome scale, focused CRISPR libraries of flexible size for specialized genetic screen purposes.