To specifically disrupt a gene is an essential tool for the investigation of its function. With the advent of CRISPR technology, it is now possible to disrupt any gene in the human genome. However, there is usually extensive functional redundancy and disruption of a single gene is often phenotypically silent. For example, out of the ca. 19,000 genes in the human genome, less than 2,000 are essential for cell proliferation. The remaining 17,000 genes are not essential or essential only in some types of cells. Characterization of their function requires the disruption of two or more genes and in multiple cell types. Yet the efficiency of CRISPR is highly variable and often low, making the isolation of successfully targeted cells a laborious task, especially when one needs to disrupt two or more genes. The goal of this application is to develop a simple plasmid DNA transfection-based method to facilitate the sequential disruption of multiple genes. It is based on the co-targeting of the hypoxanthine phosphoribosyltransferase (HPRT) gene. The basic strategy is to transfect cells with plasmids that express Cas9, a guide RNA for HPRT, and a guide RNA for the gene of interest. The HPRT mutant cells are selected with 6-thioguanine (6-TG) and are highly enriched for cells that also carry mutations in the gene of interest. The disrupted HPRT gene is then corrected by CRISPR-induced homologous recombination with a donor HPRT gene. This step is used to co-target another gene of interest. By alternating the disruption and correction of the HPRT gene, each step can be used to co-target a gene of interest, allowing one to disrupt as many genes as cell viability permits. This method requires only simple DNA transfection reagents and can achieve ca. 100% efficiency. Successful completion of the project will greatly expand the applications of the CRISPR technology to the analysis of gene function in cancer evolvement and therapy.
/RELEVANCE TO PUBLIC HEALTH The research proposed in this project will promote the improvement of public health by expanding the applications of the CRISPR technology to the analysis of gene function in cancer development and therapy.
