We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

我们开发了一种使用简单但可扩展的定向进化策略将复杂蛋白质工程化为多维规格的新方法。通过在显微镜下自动挑选被识别为同时表达多种特定特性的表达蛋白质的哺乳动物细胞，我们可以在短短几个小时内筛选出库中成千上万种蛋白质，并沿多个性能轴对每种蛋白质进行评估。为了证明这种方法的强大功能，我们创建了一个遗传编码的荧光电压指示器，同时使用我们的显微镜引导的细胞挑选策略优化了其亮度和膜的定位。我们生产了基于视蛋白的高性能荧光电压报告基因Archon1，并通过对急性小鼠脑切片和幼体斑马鱼体内的峰值和毫伏级亚阈值和突触活性进行成像，证明了其实用性。我们还测量了小鼠遗传调控神经元下游的突触后反应。秀丽隐杆线虫。