
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.03.610999
preprint
1
Article
Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells
Zouein Annalise http://orcid.org/0000-0001-7820-4159

Lende-Dorn Brittany http://orcid.org/0000-0003-4107-7648

Galloway Kate E http://orcid.org/0000-0001-7416-3193

Ellis Tom http://orcid.org/0000-0001-5392-976X

Ceroni Francesca http://orcid.org/0000-0001-7237-4982

03 9 2024
2024.09.03.610999https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.03.610999
nihpp-2024.09.03.610999.pdf
Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.
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pmc
