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Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

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Published on: March 9, 2017

Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.

Yang Liu^1,2, Xinyi Wan^1,2, Baojun Wang^3,4

¹School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FF, UK.

Nature Communications

|August 28, 2019

Summary

Researchers developed a novel CRISPR activation (CRISPRa) system for bacteria using σ⁵⁴-dependent promoters. This system offers enhanced flexibility, multi-input regulation, and high dynamic ranges for gene expression control in synthetic biology applications.

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Area of Science:

Synthetic Biology
Molecular Biology
Microbial Biotechnology

Background:

CRISPR interference (CRISPRi) is established for gene silencing in bacteria.
Existing CRISPR activation (CRISPRa) systems in bacteria have limitations in flexibility and activity due to reliance on σ⁷⁰ promoters.

Purpose of the Study:

To develop a more versatile and active CRISPRa system for bacterial gene expression control.
To enable multi-input regulation and high dynamic range gene activation in bacteria.
To create a platform for optimizing metabolic pathways and advancing synthetic biology.

Main Methods:

Development of a eukaryote-like CRISPRa system utilizing σ⁵⁴-dependent promoters.
Integration with dxCas9 for expanded DNA targeting flexibility.
Construction of cascaded CRISPRa circuits and a reusable metabolic pathway optimization platform.

Main Results:

The new CRISPRa system demonstrates high efficiency in activating relevant σ⁵⁴-dependent promoters in non-model bacteria.
Achieved long-distance, multi-input regulation with high dynamic ranges.
Enabled orthogonal gene regulation on multiple levels and facilitated construction of complex genetic circuits.
Successfully applied to create a platform for metabolic pathway optimization.

Conclusions:

The developed σ⁵⁴-dependent CRISPRa system significantly enhances bacterial gene regulation capabilities.
This system offers a powerful and versatile tool for synthetic biology, research, and industrial applications.
It overcomes limitations of previous CRISPRa systems, enabling advanced genetic engineering strategies.