Po-327 Modelling intratumor heterogeneity through CRISPR-barcodes

Abstract

IntroductionCancer is an evolutionary process in which the stepwise accumulation of genetic alterations is shaped by Darwinian selection. As a result, each tumour is composed of a complex mixture of clonal cell subpopulations containing a partially overlapping, but distinct, pattern of driver and passenger mutations. Such intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for resistance to therapy. We recently devised a novel approach based on CRISPR/Cas9 technology to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity.Material and methodsWe used CRISPR/Cas9 technology to insert a potentially functional modification in the sequence of a gene of interest, coupled to a series of silent point mutations, serving as a genetic label for cell tracing. Because of HDR low efficiency, the modified allele can be introduced only in a small fraction of cells, thus mimicking the emergence of new mutant clones within a cancer mass population. In parallel, a second barcode, consisting of different silent mutations, was used as an internal control for possible CRISPR off-target cleavage. After exposing the cells to a given selective condition, the effects of the mutation of interest can be analysed by measuring the proportion of each barcode in genomic DNA by real-time quantitative PCR or deep sequencing.Results and discussionsWe used this CRISPR-barcoding strategy to model different mechanisms of non-small-cell lung cancer resistance to EGFR inhibitors, and we established a multiplex system to evaluate the efficacy of combined drug therapies aimed at preventing or delaying the emergence of resistant cells. Through a similar approach, we assessed the effects of repairing oncogenic driver mutations in addicted cancer cells directly at the genome level. Finally, we used a highly complex set of CRISPR-barcodes to trace intratumor heterogeneity, as a convenient alternative strategy to lentiviral barcode libraries.ConclusionCRISPR-barcoding is a fast and highly flexible means to investigate the effects of different kinds of genomic modifications in a broad range of functional assays.