High-throughput, CRISPR/Cas9-activator mediated profiling of enhancer elements that drive senescence
CDKN2A is located among a hotspot of aging associated elements, and encodes two tumor suppressors, p16INK4a and p14ARF.In this project, we seek to probe the complex regulatory landscape of CDKN2A with unprecedented coverage by developing a high-throughput assay to scan large swaths of genetic regulatory regions for promoter and enhancer elements. We utilize programmable transcriptional activators (PTAs) to induce transcription of CDKN2A by expressing a library of gRNAs that target the promoter region of the gene. In our platform, we constitutively express a dCas9-based PTA and gRNA targeting the CDKN2A core promoter. Importantly, we encode a second gRNA expression cassette in the genomic region encoding the 3’ UTR of CDKN2A. This second gRNA will direct the PTA to a putative enhancer region in the 100 kb neighborhood of CDKN2A, serving as a unique barcode. gRNAs that target enhancer regions will produce more mRNA and hence more of their own barcode. We will take advantage of the irreversibility of BxbI to simultaneously engineer thousands of unique cell lines each encoding a single gRNA. We can then use next-generation sequencing to quantify the relative levels of each barcode to compare activation efficiency.