功能性 DNA 调节 CRISPR-Cas12a 传感器，用于非核酸目标的即时诊断,Journal of the American Chemical Society

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功能性 DNA 调节 CRISPR-Cas12a 传感器，用于非核酸目标的即时诊断
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2019-12-04 , DOI: 10.1021/jacs.9b09211
Ying Xiong ₁ , Jingjing Zhang ₂ , Zhenglin Yang , Quanbing Mou , Yuan Ma , Yonghua Xiong ₁ , Yi Lu

Affiliation

除了非凡的基因组编辑能力外，CRISPR-Cas 系统由于其高碱基分辨率和等温信号放大，开启了生物传感应用的新时代。然而，报道的 CRISPR-Cas 传感器主要仅用于检测核酸，对非核酸靶标的应用有限。为了充分发挥 CRISPR-Cas 传感器的潜力并拓宽其在非核酸靶标检测和定量方面的应用，我们在此报告了受功能性 DNA (fDNA) 分子（如适体和 DNA 酶）调控的 CRISPR-Cas12a 传感器对有机小分子和金属离子检测具有选择性。该传感器基于由 Cas12a 组成的 Cas12a 相关报告系统，CRISPR RNA (crRNA) 及其每端标记有荧光团和淬灭剂的单链 DNA 底物 (ssDNA-FQ)，以及可以锁定 Cas12a-crRNA 的 DNA 激活剂的 fDNA 分子，防止 Cas12a 在fDNA 靶标的缺失。fDNA 靶标的存在可以触发 DNA 激活剂的解锁，然后可以激活 Cas12a 对 ssDNA-FQ 的裂解，从而导致可通过市售便携式荧光计检测到的荧光信号增加。使用这种方法，在环境温度（25 ℃）下，使用简单快速的检测工作流程（两步，<15 分钟）定量检测了 ATP 和 Na+，使 fDNA 调控的 CRISPR 系统适用于现场测试或点-护理诊断。由于可以获得 fDNA 来识别广泛的目标，

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Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic Acid Targets

Beyond its extraordinary genome editing ability, the CRISPR-Cas system has opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic acid targets, we herein re-port CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detections. The sensor is based on the Cas12a dependent reporter sys-tem consisting of Cas12a, CRISPR RNA (crRNA) and its single stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssD-NA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na+ have been detected quan-titatively under ambient temperature (25 ℃) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recog-nize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system signifi-cantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanalytical and biomedical applications.

更新日期：2019-12-04

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