清脆的
化学
反式激活crRNA
泰特
计算生物学
生物传感器
核糖开关
适体
小分子
连接器
纳米技术
组合化学
生物物理学
Cas9
转录因子
核糖核酸
分子生物学
生物化学
抑制因子
生物
计算机科学
非编码RNA
基因
操作系统
材料科学
作者
Ahmed Mahas,Qiaochu Wang,Tin Maršić,Magdy M. Mahfouz
标识
DOI:10.1021/acs.analchem.1c04332
摘要
Cell-free biosensors can detect various molecules, thus promising to transform the landscape of diagnostics. Here, we developed a simple, rapid, sensitive, and field-deployable small-molecule detection platform based on allosteric transcription factor (aTF)-regulated expression of a clustered regularly interspaced short palindromic repeats (CRISPR) array coupled to Cas12a activity. To this end, we engineered an expression cassette harboring a T7 promoter, an aTF binding sequence, a Cas12a CRISPR array, and protospacer adjacent motif-flanked Cas12a target sequences. In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array; this leads to activation of Cas12a collateral activity, which cleaves a single-stranded DNA linker to free a quenched fluorophore, resulting in a rapid, significant increase of fluorescence. As a proof of concept, we used TetR as the aTF to detect different tetracycline antibiotics with high sensitivity and specificity and a simple, hand-held visualizer to develop a fluorescence-based visual readout. We also adapted a mobile phone application to further simplify the interpretation of the results. Finally, we showed that the reagents could be lyophilized to facilitate storage and distribution. This detection platform represents a valuable addition to the toolbox of cell-free, CRISPR-based biosensors, with great potential for in-field deployment to detect non-nucleic acid small molecules.
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