CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer. [Display omitted] •CRISPR-barcoding is a versatile method to trace clonal dynamics in tumor populations•Sensitive barcode detection allows DNA editing even when HDR efficiency is low•A silent barcode is used as a control for potential Cas9 off-target cleavage•Highly complex barcodes are used to label thousands of cells within a mass population Guernet et al. used CRISPR/Cas9 technology to genetically label and trace tumor cells within a mass population through insertion of a series of point mutations at a specific genomic location. The effects of a particular experimental condition on the fraction of barcoded cells can be assessed by qPCR or deep sequencing.