Methods for mammalian genetic analysis

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

BACKGROUND. Accurate modeling of the gene mutation and expression patterns seen in human tumors is essential for cancer research. Gene expression can be downregulated with RNAi whereas CRISPR/Cas9 can introduce precise gene mutations in mammalian cells. A major challenge in studying the cancer genome is that we can only model a small fraction of the complex changes observed in a tumor cell. Thus, developing methods that enable genome editing on a more complex scale is critical for the modeling of cancer cell behavior in pre-clinical studies. OBJECTIVES. 1) To develop combinatorial RNAi to simultaneously downregulate multiple genes in human and mouse cancer cells; 2) to develop combinatorial CRISPR/Cas9 gene knockout methods for deleting multiple genes concurrently in human and mouse cells; and 3) to establish methods and reagents for precise gene editing in human and mouse cells, enabling the introduction of mutations and the regulation of 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 siRNAs. This method enables the simultaneous suppression of the expression of up to seven genes in the same cell. This level of complexity allows us to interrogate gene paralog redundancy and gene node interaction interaction in human cancer cells. This work has been published. 2) Multiplexed CRISPR/Cas9 for combinatorial gene knockout. We have developed a multiplexed CIRSPR/Cas9 gene knockout method. This method enables the simultaneous disruption of three genes in human cancer cells. This work has been published. We are currently developing methods to knockout multiple genes in mouse and human organoids. 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 focused CRISPR libraries of flexible size for specialized genetic screens where library complexity is a limiting factor. This work has been published. 4) Development of efficient gene editing method in organoids. We are currently working on developing a method to introduce precise gene mutations in primary organoid cells from mouse. This will enable us to generate isogenic organoid cells with different combination of oncogenic mutations rapidly. These organoids can be used to test cancer drug response and tumor behavior in preclinical models.