Garimella Shruthi S, Minami Shiaki A, Khanchandani Anusha N, Abad Santos Justin C, Schaffer Susannah R, Shah Priya S
Department of Chemical Engineering, University of California, Davis, USA.
Department of Microbiology and Molecular Genetics, University of California, Davis, USA.
BMC Biotechnol. 2025 Jul 1;25(1):58. doi: 10.1186/s12896-025-00994-2.
Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling 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.
The 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. Using human HEK293T cells, 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. We also demonstrate the optimal LACE system also depends on cell type. In mouse myoblast C2C12 cells, 2pLACE displayed less variability compared to 4pLACE, similar to HEK293T cells. However, 2pLACE also had a smaller dynamic range in C2C12 cells compared to 4pLACE.
This 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. However, the optimal system may depend on cell type and application.
光遗传学系统利用光响应蛋白通过“轻触开关”来控制基因表达、离子通道、蛋白质定位和信号传导。光激活CRISPR效应器（LACE）系统就是这样一种工具。它以可调节、可逆且空间分辨的方式调节基因表达的能力使其在许多应用中具有吸引力。然而，LACE依赖于在单个质粒上递送四个单独的组件，这可能会限制其使用。在此，我们对LACE进行优化，以减少递送所有四个组件所需的质粒数量。
双质粒LACE（2pLACE）系统将原始LACE系统的四个组件组合到两个质粒中。构建完成后，以增强型绿色荧光蛋白（eGFP）表达的光遗传学控制作为报告基因，对2pLACE的行为进行了严格测试。我们使用人胚肾293T细胞，优化了两个质粒的比例，测量了激活作为光强度的函数，并确定了激活最大荧光的光频率。总体而言，2pLACE系统显示出与原始的四质粒LACE（4pLACE）系统相似的动态范围、可调性和激活动力学。有趣的是，与4pLACE相比，2pLACE在激活信号方面的变异性也较小。我们还证明了最佳的LACE系统也取决于细胞类型。在小鼠成肌细胞C2C12细胞中，与4pLACE相比，2pLACE表现出较小的变异性，与293T细胞相似。然而，与4pLACE相比，2pLACE在C2C12细胞中的动态范围也较小。
这种简化的光遗传学系统将更适合需要将变异性最小化的生物技术应用。通过优化LACE系统以使用更少的质粒，2pLACE成为多种研究应用中的灵活工具。然而，最佳系统可能取决于细胞类型和应用。
