Engineering digitizer circuits for chemical and genetic screens in human cells.
Engineering digitizer circuits for chemical and genetic screens in human cells.
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DOI：
10.1038/s41467-021-26359-9
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发表时间：
2021-10-22
影响因子：
16.6
通讯作者：
Wong WW
中科院分区：
文献类型：
--
作者：
Wong NM;Frias E;Sigoillot FD;Letendre JH;Hild M;Wong WW
基于细胞的转录报告基因在高通量化合物和CRISPR筛选中是非常宝贵的，用于鉴定可以影响感兴趣途径的化合物或基因。然而，许多转录报告基因的活性较弱，反应短暂。这可能导致忽略难以检测的治疗靶标和化合物，从而需要在不同时间点运行多个筛选的资源消耗过程。在这里，我们提出了雷达，一个数字化电路放大记者的活动和保留记忆的途径激活。在AP-1通路的报告中，我们的电路识别出具有已知活性的化合物对抗PKC相关通路，并显示出与化合物筛选中的经典报告者相比具有提高的灵敏度的增强的动态范围。在AP-1通路的第一个全基因组合并CRISPR筛选中，RADAR从MAPK和PKC通路中识别出典型基因，以及非典型调节因子。因此，我们的可扩展系统突出了在大规模基于细胞的筛选中使用遗传电路的益处和多功能性。基于细胞的转录报告基因是高通量筛选的宝贵组成部分，但许多此类报告基因的反应较弱或短暂。在这里，作者描述了一种数字化电路，用于放大报告活性，增加灵敏度，并保留通路激活的记忆。
Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening. Cell-based transcriptional reporters are an invaluable part of highthroughput screening, but many such reporters have weak or transient responses. Here, the authors describe a digitizer circuit for amplifying reporter activity, increasing sensitivity, and retaining memory of pathway activation.
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影响因子：
16.6
作者：
Donahue, Patrick S.;Draut, Joseph W.;Leonard, Joshua N.
通讯作者：
Leonard, Joshua N.
DOI：
10.1073/pnas.85.13.4628
发表时间：
1988-07-01
影响因子：
11.1
作者：
GATES, CA;COX, MM
通讯作者：
COX, MM
影响因子：
5.7
作者：
Beal, Jacob
通讯作者：
Beal, Jacob
影响因子：
11.4
作者：
HIRAI, S;IZUMI, Y;OHNO, S
通讯作者：
OHNO, S
影响因子：
4.8
作者：
Kamakura, S;Moriguchi, T;Nishida, E
通讯作者：
Nishida, E