PROJECT SUMMARY The overall goal of this proposal is to systematically characterize the role of transcription factor isoforms by leveraging high-throughput experimental genomics approaches. Transcription factors (TFs) are master regulators of gene expression and as such play key roles in a variety of biological processes, including cell growth and differentiation, organismal development, and response to environmental stimuli. The human genome is estimated to harbor ~1600 TF genes; however, most of these ~1600 TFs are expressed as a series of protein isoforms encoded by alternatively spliced mRNAs arising from the same locus. Though a handful of alternative TF isoforms are known to play functionally important (and distinct) roles in the cell, the overwhelming majority—thousands of proteins—remain entirely uncharacterized, and new TF isoforms continue to be discovered. Thus, decoding the roles of TF isoforms is key to a systems-level understanding of gene regulation. Here, I aim to decode the functions of TF isoforms by leveraging the novel RNA-targeting CRISPR/Cas13d system. Cas13d has recently emerged as a precise, programmatic, and efficient enzyme to use for systematic knockdown of RNA—overcoming many of the limitations exhibited by existing approaches to perturb isoforms en masse. I will employ Cas13d to knock down thousands of TF isoforms in a single experiment, linking, for the first time, cellular phenotypes to TF isoforms, genome-wide. I will use breast cancer as a model system, as cellular phenotypes such as cell growth are highly biologically relevant to cancer, and a handful of alternative TF isoforms have been shown to play important roles in breast cancer. In Aim 1, I will establish a framework for isoform-specific knockdowns using Cas13d. I will develop an algorithm to programmatically design efficient, isoform-specific Cas13d guide RNAs, and validate them using targeted, singleplex knockdown experiments in human cell lines. In Aim 2, I will systematically assess the effects of TF isoforms on cellular growth, using breast cancer as a model system. I will perform a Cas13d-based pooled screen to identify TF isoforms—both annotated and unannotated—that play biologically important roles in breast cancer cell growth. By completing this proposal, I will develop novel technologies that can be employed to move beyond a rigid “gene-centric” framework and towards an “isoform- level” framework, which more accurately captures the deep complexity encoded in the human genome. Moreover, I will shed light on the role that TF isoforms play in breast cancer, which will prioritize candidates for future mechanistic studies. During this Fellowship, I will further refine my expertise in bioinformatics while complementing it with new training in experimental, high-throughput functional genomics. Ultimately, I aim to run my own independent research group that employs a combination of computational and experimental approaches to probe the mysteries of the ...