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

Abstract

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.

Applications of Serine Integrases in Synthetic Biology over the Past Decade

“…An impressive work by Kasari et al reported the decoupling of E. coli growth and production by removing the chromosomal origin of replication (relying on the phiC31 expression under the control of temperature-sensitive cI857 repressor), which caused the elongation phenotype of E. coli as filaments [102]. E. coli cells could also become spheres by removing the mreB gene [103]. Another phenotype change is cell differentiation.…”

BatCRISPRi: Bacillus titratable CRISPRi for dynamic control inBacillus subtilis

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