Methods for mammalian genetic analysis

背景资料。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的模式。RNAi可下调基因表达。最近，CRISPR/Cas9已成为在哺乳动物细胞中引入突变和改变基因表达的主要基因组编辑工具。对癌症基因组建模的一个主要挑战是，我们目前只能概括在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将对临床前研究中的癌细胞行为建模至关重要。目标。1)建立在人和小鼠细胞中同时下调多个基因表达的组合RNAi方法和试剂；2)建立在人和小鼠细胞中同时删除多个基因的组合CRISPR/Cas9基因敲除方法和试剂；3)开发在人和小鼠细胞中引入突变和调节基因表达的基因编辑方法和试剂。重大活动、重大成果、关键成果。1)。用于组合基因敲除的多重RNAi。我们开发了一种使用高效siRNAs的多重基因敲除方法。这种方法可以在同一细胞中同时敲除多达7个基因。这种程度的复杂性使我们能够询问人类癌细胞中的基因平行冗余和多个遗传节点的功能相互作用。这部作品出版了。2)。多重CRISPR/Cas9用于组合基因敲除。我们已经开发了一种使用预先验证的合成引导RNA的多路CIRSPR/Cas9基因敲除方法。这种方法能够同时敲除人类癌细胞中的多达3个基因。这种方法可以在多个基因座位上进行快速基因组编辑。这部作品出版了。3)。用于遗传筛查的模块化CRISPR文库的开发。我们已经开发了一个汇集sgRNAs库模块的精选集合。每个模块中的基因都由它们的功能注释来管理。这使得构建亚基因组规模、具有灵活大小的聚焦CRISPR文库用于专门的遗传筛选目的成为可能。

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.