In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 120, No. 30 ( 2023-07-25)
Abstract:
Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs 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) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli -based cell-free system. These components enable CRISPRa GRNs 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 GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.2220358120
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2023
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
