A simplified two-plasmid system for orthogonal control of gene expression using light-activated CRISPR effector for mammalian cells

Garimella, S. S.; Minami, S. A.; Khanchandani, A. N.; Schaffer, S. R.; Shah, P.

doi:10.1101/2024.12.13.628044

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bioRxiv · 10.1101/2024.12.13.628044

A simplified two-plasmid system for orthogonal control of gene expression using light-activated CRISPR effector for mammalian cells

Garimella, S. S.·Minami, S. A.·Khanchandani, A. N.·Schaffer, S. R.·Shah, P.

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Abstract

BackgroundOptogenetic systems use light-responsive proteins to control gene expression with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components. ResultsThe two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. We optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. ConclusionsThis simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications.

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Garimella, S. S., Minami, S. A., Khanchandani, A. N., Schaffer, S. R., Shah, P.. 2024-12-16. A simplified two-plasmid system for orthogonal control of gene expression using light-activated CRISPR effector for mammalian cells. https://doi.org/10.1101/2024.12.13.628044

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