Generation and tracking of cells with precise edits

Current methods of genetically-engineered cells require precise integration of genetic material. Genotyping is usually used to detect genetic edits; however, it's difficult to track the exact changes made in each individual cell in a large cellular mixture, especially when the cells receive multiple edits. While whole genome genotyping is available to track each cell in a mixture, the technique is expensive and time-consuming. Thus, there is a need for additional methodologies that enables quicker and cost-efficient tracking of genetic edits within cells of interest.

The Fraser lab at Stanford has invented a genetic engineering platform that enables multiple cellular gene editing while tracking the exact edits being made. The invention consists of two or more CRISPEY modules covalently produced by the vector, where the combination of edits that will be made across all modules are predetermined. Each module applies edits of interest tagged with unique sequences that can be easily genotyped by DNA sequencing or other methods. When needed, the intended combination of precise edits for each can also be inferred by Sanger sequencing, NGS, or other detection methods. Unlike existing methods, the invention can be applied to a mixture of cells as well as single cell or clonal cell lineages, and provides an efficient, high-throughput, and low-cost method for genetic and cellular engineering.