Methods for mammalian genetic analysis

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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.