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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.
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This content will become publicly available on December 1, 2026
Scoping chromatin dynamics across cortical neurogenesis
High-resolution profiling of dynamic gene regulatory changes during brain development remains challenging. In this issue of Neuron, Jia, Hou, Cui, Luo, and Duan et al. present SCOPE-C, a low-input method to simultaneously capture open chromatin and dynamic cis-regulatory contacts, revealing cell- type-specific and human-biased regulatory hubs during corticogenesis.
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- Award ID(s):
- 2145885
- PAR ID:
- 10654929
- Publisher / Repository:
- Cell Press
- Date Published:
- Journal Name:
- Neuron
- Volume:
- 113
- Issue:
- 23
- ISSN:
- 0896-6273
- Page Range / eLocation ID:
- 3881 to 3883
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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