Curated loci prime editing (cliPE) to measure variant effects in TSC2

Tuberous sclerosis is caused by loss-of-function variants in two genes, TSC1 and TSC2. Missense variants in TSC2 are often classified as variants of uncertain significance in clinical genetic testing for tuberous sclerosis or cancer risk. Prior work has established that assays measuring the activity of TSC2 in heterologous systems can aid variant classification. We used curated loci prime editing (cliPE) to generate pools of haploid cells with genetic variants in TSC2. These cell pools were serum starved overnight, fixed, and labeled for phosphorylated S6 (pS6), a well characterized biomarker of mTOR pathway activity. Cells were sorted using fluorescence activated cell sorting and genomic DNA was extracted from cells with high levels of pS6 and unsorted cells. PCR of the prime edited regions was used to generate amplicon sequencing libraries. Variant allele frequencies can be compared between unsorted and sorted cells to determine a variant-specific TSC2 activity score.