Abstract C162: A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality (2015)

Abstract Target selection for oncology and other indications is a critical step in the successful development of therapeutics, however it remains one of the most challenging areas of drug discovery. In fact, up to two-thirds of oncology relevant targets reported in literature have not been confirmed on follow-up studies, indicating that target validation in oncology is especially challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of specific loci offers an alternative method to RNA interference and complements small molecule inhibitors for determining whether or not a cell line is dependent on a specific gene product for proliferation and/or survival. Importantly, CRISPR-Cas9 may be advantageous for some studies as it offers efficient and specific gene knock-out leading to complete loss of protein function. This may be especially useful for some target classes, including epigenetic targets, which appear to require near complete loss of protein function to observe phenotypes. In our initial studies using CRISPR-Cas9 to verify the essential nature of EZH2 (Enhancer of Zest 2) expression for the proliferation of SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor cell lines, we observed that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity at least one allele of EZH2 remains functional, and this hypothesis suggests that carrying out CRISPR-Cas9 studies for individual target genes without analyzing single cell clones could produce misleading results. To verify this, we developed a medium throughput assay to analyze 10s-100s of single cell clones for target gene disruption using CRISPR-Cas9 gene knockout, followed by a restriction digest and fluorescent undigested fragment length analysis to successfully assess EZH2 allele status. Significantly, these data support our hypothesis that retention of one functional copy of EZH2 is required for the proliferation of EZH2-dependent cell lines and that this can be rapidly assessed by our assay. Thus, the assessment of zygosity of the gene of interest can be evaluated in a medium-throughput manner in single cell clones. The assay is thereby able to unambiguously indicate whether or not a specific gene is essential for survival and/or proliferation in a given cell line, and offers a unique approach for target validation using gene editing. Importantly, this approach should be applicable to any target of interest that is expected to affect cell proliferation or survival. Such data can aid in the development of more robust cancer therapeutics by increasing confidence in target selection. Citation Format: Alexandra R. Grassian, Tim Scales, Sarah K. Knutson, Nicola J. McCarthy, Chris E. Lowe, Jon D. Moore, Robert A. Copeland, Heike Keilhack, Jesse J. Smith, Julie A. Wickendon, Scott Ribich. A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C162.