American Chemical Society
sb3c00035_si_002.avi (173 MB)

OptoCRISPRi-HD: Engineering a Bacterial Green-Light-Activated CRISPRi System with a High Dynamic Range

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posted on 2023-05-23, 01:44 authored by Ke-Ning Chen, Bin-Guang Ma
The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space–time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.