Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.
Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.
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光遗传学微孔阵列筛选系统：基因编码荧光指示剂的高通量工程平台。
DOI：
10.1021/acssensors.3c01573
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发表时间：
2023
期刊：
影响因子：
8.9
通讯作者：
Berndt,Andre
中科院分区：
文献类型：
--
作者：
Rappleye,Michael;Wait,SarahJ;Lee,JustinDaho;Siebart,JamisonC;Torp,Lily;Smith,Netta;Muster,Jeanot;Matreyek,KennethA;Fowler,DouglasM;Berndt,Andre
遗传编码荧光指示器（gefi）是一种基于蛋白质的光遗传学工具，当与细胞和组织中的特定配体结合时，可以改变其荧光强度。编码DNA的GEFI可以在细胞亚型中表达，同时监测细胞的生理反应。然而，具有生理敏感性和药理学特异性的工程gefi在逐个评估其生物物理功能的同时，往往需要通过试错诱变进行反复优化。在这里，蛋白质的巨大突变景观构成了减缓GEFI发展的重大障碍，特别是对于依赖哺乳动物宿主系统进行测试的传感器。为了克服这些障碍，我们开发了一种多路复用的高通量工程平台，称为光遗传微孔阵列筛选系统（optomass），该系统可以在哺乳动物细胞中并行测试数千种GEFI变体。Opto-MASS代表了工程光遗传工具的下一步，因为它可以比现有方法更快地筛选大型变体库。我们通过测试超过13000种多巴胺和21000种阿片传感器变体来展示这个系统。我们生成了一个新的多巴胺传感器dMASS1，与母体结构相比，当多巴胺暴露在100 nM时，其信号增加了60倍。我们的新型阿片传感器μMASS1比其母体支架对500 nM DAMGO的响应增加了约4.6倍的信号。因此，Opto-MASS可以快速设计新的传感器，同时显著缩短具有不同生物物理性质的新传感器的优化时间。
Genetically encoded fluorescent indicators (GEFIs) are protein-based optogenetic tools that change their fluorescence intensity when binding specific ligands in cells and tissues. GEFI encoding DNA can be expressed in cell subtypes while monitoring cellular physiological responses. However, engineering GEFIs with physiological sensitivity and pharmacological specificity often requires iterative optimization through trial-and-error mutagenesis while assessing their biophysical functionin vitroone by one. Here, the vast mutational landscape of proteins constitutes a significant obstacle that slows GEFI development, particularly for sensors that rely on mammalian host systems for testing. To overcome these obstacles, we developed a multiplexed high-throughput engineering platform called the optogenetic microwell array screening system (Opto-MASS) that functionally tests thousands of GEFI variants in parallel in mammalian cells. Opto-MASS represents the next step for engineering optogenetic tools as it can screen large variant libraries orders of magnitude faster than current methods. We showcase this system by testing over 13,000 dopamine and 21,000 opioid sensor variants. We generated a new dopamine sensor, dMASS1, with a >6-fold signal increase to 100 nM dopamine exposure compared to its parent construct. Our new opioid sensor, μMASS1, has a ∼4.6-fold signal increase over its parent scaffold’s response to 500 nM DAMGO. Thus, Opto-MASS can rapidly engineer new sensors while significantly shortening the optimization time for new sensors with distinct biophysical properties.
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影响因子：
7.3
作者：
Hackley CR;Mazzoni EO;Blau J
通讯作者：
Blau J
DOI：
10.1126/science.aat4422
发表时间：
2018-06-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Patriarchi T;Cho JR;Merten K;Howe MW;Marley A;Xiong WH;Folk RW;Broussard GJ;Liang R;Jang MJ;Zhong H;Dombeck D;von Zastrow M;Nimmerjahn A;Gradinaru V;Williams JT;Tian L
通讯作者：
Tian L
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
8.6
作者：
Erdogan, Mutlu;Fabritius, Arne;Griesbeck, Oliver
通讯作者：
Griesbeck, Oliver
影响因子：
16.6
作者：
Kroning, Kayla E.;Wang, Wenjing
通讯作者：
Wang, Wenjing