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Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor
oleh: Yufeng Zhao, Sungmoo Lee, Robert E. Campbell, Michael Z. Lin
Format: | Article |
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Diterbitkan: | MDPI AG 2023-05-01 |
Deskripsi
The use of genetically encoded fluorescent sensors for the calcium ion (Ca<sup>2+</sup>) has revolutionized neuroscience research by allowing for the recording of dozens of neurons at the single-cell level in living animals. However, fluorescence imaging has some limitations such as the need for excitation light, which can result in a highly auto-fluorescent background and phototoxicity. In contrast, bioluminescent sensors using luciferase do not require excitation light, making them ideal for non-invasive deep tissue imaging in mammals. Our lab has previously developed a bioluminescent Ca<sup>2+</sup> sensor CaMBI to image Ca<sup>2+</sup> activity in the mouse liver, but its responsiveness to Ca<sup>2+</sup> changes was suboptimal. To improve the performance of this sensor, we applied directed evolution to screen for genetic variants with increased responsiveness. Through several rounds of evolution, we identified variants with more than five times improved responsiveness in vitro. We characterized the improved sensors in culture cell lines and dissociated rat neurons and confirmed that they exhibited a higher sensitivity to changes in intracellular Ca<sup>2+</sup> levels compared to their progenitor. These optimized Ca<sup>2+</sup> sensors have the potential for non-invasive imaging of Ca<sup>2+</sup> activity in vivo, particularly in the brain.