Abstract PR10: Functional analysis of oncogenic driver mutations in human cancer cells through CRISPR-barcoding

Abstracts: Fourth AACR International Conference on Frontiers in Basic Cancer Research; October 23-26, 2015; Philadelphia, PA Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been recently engineered into a new DNA editing technology that enables to custom modify the genome of virtually any type of cells. In the last three years, CRISPR has been used for a wide array of applications, including the generation of genetically modified organisms and new cancer models, as well as for functional studies in cultured cells. However, despite the extraordinary possibilities offered by CRISPR, two major potential drawbacks need to be considered when using this technology: the possibility of off-target DNA editing and the necessity to derive individual clones containing the desired genetic modification, which implies a relatively high level of efficiency. To overcome these intrinsic limitations, we have devised CRISPR-barcoding, a new strategy in which a potentially functional modification in the sequence of a gene of interest is coupled with a series of silent point mutations, functioning as a genetic label for cell tracking. In parallel, a second barcode consisting of distinct silent mutations is inserted in the same cell population and used as a control for CRISPR off-target effects. The genomic DNA from the resulting mixture of CRISPR-modified and unmodified cells is then probed by real-time quantitative PCR using specific primers to assess the relative proportion of each barcode. Hence, by exposing the cells to a given selective condition, this approach can be used to functionally characterize the effects of different types of mutations of a particular gene of interest. As a proof-of-concept of our strategy, we repaired the mutated sequence of the tumor suppressor APC in colorectal cancer cells, and showed that APC restoration resulted in the inhibition of both Wnt signaling reporter activity and cell growth. To further illustrate the wide range of potential applications of this approach, we used CRISPR-barcoding to modify the sequence of other cancer-related genes, including TP53, ALK, EGFR and KRAS, in various tumor cells, and we assessed the effects of such alterations on cell growth, invasion and resistance to chemotherapy, both in vitro and in vivo. Thus, by preventing the limitations associated with the low efficiency and the potential off-target effects of DNA editing, our studies demonstrate that CRISPR-barcoding is a convenient and effective strategy to investigate the functional consequences of a specific genetic modification. Citation Format: Alexis Guernet, Dorthe Cartier, Sathish Kumar Mungamuri, Sahil Adriouch, Myriam Vezain, Francoise Charbonnier, Sophie Coutant, Shen Yao, Hassan Ainani, David Alexandre, Olivier Boyer, Stuart A. Aaronson, Youssef Anouar, Luca Grumolato. Functional analysis of oncogenic driver mutations in human cancer cells through CRISPR-barcoding. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR10.