Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators that regulate these key cellular programs in CRC, we developed an endogenous reporter system by genome-editing human CRC cell lines with knock-in fluorescent reporters at the SOX9 and KRT20 locus to report aberrant stem cell-like activity and differentiation, respectively, and then performed pooled genetic perturbation screens. Constructing a dual reporter system that simultaneously monitored aberrant stem cell-like and differentiation activity in the same CRC cell line improved our signal to noise discrimination. Using a focused-library CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs, we identified factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency in CRC and required for in vivo growth of human CRC models. These studies highlight the utility of a biologically designed endogenous reporter system to uncover novel therapeutic targets for drug development.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics 1-3 . Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs 4-6 . The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans -labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4 DCAF16 ligase to the second bromodomain of BRD4 (BRD4 BD2 ). BRD4 BD2 , in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4 BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16 Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4 BD2 revealed that the loop conformation around BRD4 His437 , rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.