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. 2023 May 25;19(7):887–899. doi: 10.1038/s41589-023-01344-z

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a, Network diagram of regulatory interactions and phenotypic impacts for native and ectopic BACH1. ‘BACH1e’ indicates the ectopic BACH1 gene introduced and controlled via the mNF gene circuit, whereas ‘BACH1n’ indicates the native BACH1 gene; ‘BACH1p’ indicates the BACH1 protein. The same notation applies to RKIP. b, A phenotype as a function of the mean and/or variability of one or more protein’s levels defines a phenotypic landscape. Conventional therapy assumes monotone phenotypic landscapes. However, uncovering the actual landscape requires protein-level tuning. c, Schematic diagram of the two-step strategy for repeatable AAVS1 site-specific integration of genetic payloads, such as synthetic gene circuits. Left: LP insertion into AAVS1 with CRISPR–Cas9. Right: RMCE-based LP-specific integration of the mNF gene circuit that controls the expression of the eGFP::BACH1 bifunctional fusion. Step 2 is repeatable by using different selection markers. espCas9, enhanced specificity SpCas9; NeoR, neomycin resistance gene. d, Synthetic mNF gene circuit for dox-controlled tuning of the TetR co-expressed with either the GFP reporter (mNF-GFP) protein levels or GFP::BACH1 fusion (mNF-BACH1) protein levels after site-specific integration. KS, Kozak sequence; TetO, tetracycline operator.