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bioRxiv · 10.1101/2025.06.15.659618

Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation

Gu, B.·Linton, J.·Hendrickson, B. G.·Li, H.·Hadas, R.·Manella, G.·Gregrowicz, J.·Elowitz, M. B.
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Abstract

The Transforming Growth Factor beta (TGF-{beta}) 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-{beta} receptor profiles, by knocking down different receptor subsets, revealed functional interactions between the canonical BMP and TGF-{beta} 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-{beta} superfamily collaboratively process signals, and establish Pathway Sculptor as a general platform for high-order combinatorial perturbation.

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Gu, B., Linton, J., Hendrickson, B. G., Li, H., Hadas, R., Manella, G., Gregrowicz, J., Elowitz, M. B.. 2025-06-16. Pathway Sculptor for Compact and Versatile Combinatorial Genetic Perturbation. https://doi.org/10.1101/2025.06.15.659618

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