Integrating Chemical Genetic Approaches with Precision Genome Editing

Project Summary Chemical genetic approaches are powerfully enabling for biological discovery and therapeutics development. The identification of mutant alleles that enhance or suppress the activity of chemical probes may not only validate on-target mechanism but also drive deeper understanding of a small molecule’s binding interactions, molecular mechanism of action, and downstream biological effects. Recent breakthroughs in genome editing technologies enable the systematic mutation of endogenous proteins at scale and directly in cells, opening new research paradigms for chemical genetic approaches. Exploiting these new technologies, our prior work helped pioneer the development of in situ CRISPR-mutational scanning approaches to systematically profile protein target(s) sequence-function relationships in their native cellular environment. When leveraged with chemical biology, these mutations in the target can be exploited as discovery tools to study small molecule mechanism of action and target biology, allowing us to uncover mechanisms of allosteric regulation, cell signaling, and cancer vulnerabilities. This chemical genomics platform allows us to unlock the serendipitous discoveries that both chemical probes and cutting-edge genetic screens can afford. To push the boundaries of chemical genomics, these approaches and their associated tools will need to be innovated, expanded, and tested in biologically meaningful systems and contexts that address key questions in the field. Hence, this proposal aims to significantly broaden the scope and utility of in situ CRISPR-mutational scanning in the context of highlighting its potential to transform many facets of chemical biology: to (1) interrogate drug-target structure- function in primary cells, (2) integrate massively parallel single-cell multi-omic readouts of mutant phenotypes, and (3) illuminate the function, mechanisms, and biology of protein disordered regions as well as (4) multi-subunit protein complexes. We focus our future studies on (1) androgen receptor, a critical cancer gene, where integrating high-throughput, direct readouts of transcription factor function will be transformative, and (2) components involved in targeted protein degradation, where holistically interrogating the ubiquitin-proteasome system at scale is essential for understanding the whole pathway. Across these aims, the biology and mechanisms of various protein complexes will be deeply explored by leveraging mutant alleles with cell, molecular, computational, and structural biology. Altogether, accomplishment of these objectives will advance research paradigms for chemical genomics and will further illuminate fundamental mechanisms of small molecule action and protein complex function.