PM-11 * DEVELOPMENT OF A NOVEL GENE TARGETING AND CLONE SCREENING PLATFORM TO ENGINEER COMMON PEDIATRIC GLIOMA MUTATIONS INTO MODEL CELL LINES

Abstract

The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using donor DNA templates can be associated with low yields of single cell clones harboring homozygous versus heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process, and thus better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers. To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including the Lys27Met missense mutation within histone variant H3.3 (H3F3A), and truncating mutations within protein phosphatase 1D (PPM1D) exon 6. Using our HRM protocol, both heterozygous point mutations in H3F3A, and indels in PPM1D were detectable and grouped distinctly from wild-type samples. These findings were validated using conventional Sanger sequencing and topo cloning, which confirmed that this approach could differentiate between mutant and wild-type clones. Multiple clones were generated in under four weeks using this process-flow. These findings highlight the utility and power of our unique approach to rapidly create engineered mutant cell lines. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future.