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Title: Engineering activatible promoters for scalable and multi-input CRISPRa/i circuits
Dynamic, multi-input gene regulatory networks are ubiquitous in nature. Multi-layer CRISPR-based genetic circuits hold great promise for building gene regulatory networks akin to those found in naturally-occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) gene regulatory networks. By integrating sequence-based design and in-vivo screening, we engineer activatable promoters that achieve up to 1000-fold dynamic range in an E. coli-based cell-free system. These new components enable CRISPRa gene regulatory networks that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i gene regulatory networks, including feedback loops, logic gates, multi-layer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables new classes of gene regulatory functions in cell-free systems.  more » « less
Award ID(s):
1844152 2032794
NSF-PAR ID:
10426990
Author(s) / Creator(s):
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
ISSN:
1091-6490
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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