Programmable pathway profiles reveal signaling principles of TGF-β superfamily receptors

The Transforming Growth Factor beta (TGF-β) superfamily, like other biological pathways, relies on families of co-expressed, partially redundant protein components, such as receptor subunits. The inability to systematically modulate multi-gene component expression profiles has made it difficult to understand how components and sets of components collectively process information. To overcome this, we developed Pathway Sculptor , a dCas12a-based epigenetic editing system that achieves simultaneous same-cell knockdown of at least twelve target genes. Programming TGF-β receptor profiles, by knocking down different receptor subsets, revealed functional interactions between the canonical BMP and TGF-β pathway branches. Unexpectedly, signaling within each branch depended on receptors in the opposite branch. Further, different receptor subsets played distinct roles: ACVR-class receptors modulated signaling magnitude, whereas BMPRs and TGFBRs discriminated among ligand variants. These results show how the two branches of the TGF-β superfamily collaboratively process signals, and establish Pathway Sculptor as a general platform for high-order combinatorial perturbation.