Estrogen receptor alpha is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

• PMID: 38511482
• DOI full text
• PMC full text
• PubMed

Reference Type

Journal Article

Authors

Eerlings R, Gupta P, Lee XY, Nguyen T, El Kharraz S, Handle F, Smeets E, Moris L, Devlies W, Vandewinkel B, ... Show all

Primary Lit For

Additional Lit For

Review For