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 human genome and their roles in development and disease.