Abstract 2470: Comprehensive functional modeling of pan-cancer risk SNP rs6983267 in human CRC cells via scarless CRISPR/Cas9 genome editing
Authors: Coggins Nicole B. , O'Geen Henriette , Segal David J. , Carvajal-Carmona Luis G. 
Published: September 2020

Abstract
With the continued emergence of Genome-Wide Association study-identified risk loci for complex diseases as well as a sharp increase in the number of variants of uncertain significance (VUS) identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Our lab has previously published a novel variant modeling pipeline which combines CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer variants in ~1 month. In this study, we utilize our pipeline to model risk Single Nucleotide Polymorphism (SNP) rs6983267 in human colorectal cancer (CRC) cells and present the first functional characterization for this risk SNP at the most precise level possible with current technology, isogenic clones of the same cell lines that only differ by a single base. Rs6983267 is located within a known Mycenhancer and has been shown to exert risk-allele specific increase in enhancer activity. However, we do not observe risk-allele specific upregulation of Mycexpression in our isogenic models. Instead, we find that presence of the risk allele confers significant effects within the cell locally, modulating activity of adjacent binding site for WNT transcription factor, TCF7L2, and a subset of genes associated with enhancer function. Through 3D interaction analysis, we identify difference in interactions incisacross the three genotypes including the loss of interaction with Mycin the heterozygous and homozygous reference lines, indicating genotype-specific changes of enhancer gene targets in CRC cells. Together, these variations lead to global effects, affecting transcriptional expression of genes involved in WNT signaling, cell-to-cell adhesion and PI3K-Akt signaling pathways. These results suggest the loss of a single risk allele in CRC cells confers a transcriptional change associated with overall decreased cancer activity despite expression of the putative target gene of the enhancer, Myc, not being significantly affected. Interestingly, engineered loss of both risk alleles in CRC cells does not further the overall effect, indicating the activation of a compensatory mechanism, possibly due to redundancy of regulatory pathways affected. Activation of such compensatory mechanisms could demonstrate an important limitation to the use of established cancer cell lines in the functional modeling of risk-associated variants, which are the current standard models in the field. Our study provides an important contribution to the current efforts of functional characterization of novel VUSs and non-coding SNPs associated with cancer risk. It will become important to consider validation standards and functional thresholds for inclusion of lower penetrance susceptibility alleles in clinical decision-making as the field continues to advance towards the ultimate goal of precision medicine in cancer.
Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Comprehensive functional modeling of pan-cancer risk SNP rs6983267 in human CRC cells via scarless CRISPR/Cas9 genome editing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2470.