1.
Enhanced or reversible RNA N6-methyladenosine editing by red/far-red light induction.
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Tang, H
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Han, S
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Jie, Y
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Jiang, X
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Zhang, Y
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Peng, J
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Wang, F
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Li, X
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Zhou, X
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Jiang, W
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Weng, X
Abstract:
The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine.
2.
A dual light-controlled co-culture system enables the regulation of population composition.
Abstract:
With the development of metabolic engineering, increasing requirements for efficient microbial biosynthesis call for establishment of multi-strain co-culture system. Dynamic regulation of population ratios is crucial for optimizing bioproduction performance. Optogenetic systems with high universality and flexibility have the potential to realize dynamic control of population proportion. In this study, we utilized an optimized chromatic acclimation sensor/regulator (CcaS/R) system and a blue light-activated YF1-FixJ-PhlF system as induction modules. A pair of orthogonal quorum sensing systems and a toxin-antitoxin system were employed as communication module and effector module, respectively. By integrating these modules, we developed a dual light-controlled co-culture system that enables dynamic regulation of population ratios. This co-culture system provides a universal toolkit for applications in metabolic engineering and synthetic biology.
3.
Development of Optogenetic Dual-Switch System for Rewiring Metabolic Flux for Polyhydroxybutyrate Production.
Abstract:
Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.
