Abstract 1445: Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract

Abstract Colorectal Cancer (CRC) is one of the leading causes of mortality in the US, being the third most common cancer among both men and women. While an exact cause of CRC has not yet been elucidated, the existence of a clear heritable genetic component has been established. With the recent surge of data from Genome-Wide Association Studies, a handful of CRC-associated loci have been identified, each containing numerous Single Nucleotide Polymorphisms (SNPs). We hypothesize that among the ~25 loci associated with increased risk for CRC, there are driver SNPs functioning to contribute to initiation or progression of CRC development. It is crucial, in order to enhance our understanding CRC tumorigenesis, to extract these functional variants from the pool of associated SNPs and validate their causative significance within the cell. To model these functional SNPs, we have developed a novel “scarless” isolation pipeline for selecting isogenic clones with the desired single-base edit without selection markers following CRISPR-Cas9 mediated HDR. With this pipeline, we have successfully modeled our first selected SNP, rs6983267, from the 8q24 risk locus in HCT-116 cells, producing the heterozygous genotype (G/T) from the parental homozygous risk (G/G) genotype. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in CRC pathogenesis. In line with previous studies, we observe a slight decrease in c-Myc expression in our heterozygous clones compared to wildtype homozygous risk. Moreover, we observe a preferential binding of the transcription factor in control of c-Myc transcriptional regulation, TCF7L2, to the risk allele over the reference allele while binding of proximal factors such as CTCF show no allelic preference. Interestingly, we also see significant changes in expression of target genes of the WNT signaling pathway. Dysregulation of WNT is a hallmark characteristic of CRC. These changes indicate there is indeed an effect of the risk allele of rs6983267 on overall WNT activity in CRC cells due to increased binding of transcription factor TCF7L2. This novel SNP editing pipeline provides a method to study precise effects of single base changes located anywhere in the genome. Utilizing it to model other risk SNPs will enable the enhancement of our current understanding of GWAS-identified risk loci in CRC pathogenesis at the molecular level. Citation Format: Nicole Coggins, Jacob Stultz, David Segal, Luis Carvajal-Carmona. Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2017-1445