A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
复制标题
一种用于超灵敏 DNA 检测的 CRISPR-Cas9 触发链置换扩增方法。
DOI：
10.1038/s41467-018-07324-5
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发表时间：
2018-11-27
影响因子：
16.6
通讯作者：
Yu, Xue-Feng
中科院分区：
文献类型：
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作者：
Zhou, Wenhua;Hu, Li;Yu, Xue-Feng
虽然聚合酶链反应（PCR）是最广泛使用的DNA扩增方法，但热循环的要求限制了其非实验室应用。因此，等温DNA扩增技术代替传统PCR用于现场诊断应用是有价值的。在这里，我们描述了一种基于CRISPR-Cas9触发的切口核酸内切酶介导的链置换扩增方法（即CRISDA）的用于扩增和检测双链DNA的真正等温方法。CRISDA利用CRISPR效应子的高灵敏度/特异性和独特的构象重排来识别靶DNA。与肽核酸（PNA）侵入介导的终点测量相结合，该方法在复杂样品背景下检测各种DNA靶标时表现出阿托摩尔灵敏度和单核苷酸特异性。此外，通过将该技术与Cas9介导的靶富集方法相结合，CRISDA表现出亚阿摩尔灵敏度。总之，CRISDA是一种功能强大的等温工具，可用于即时诊断和现场分析中的核酸超灵敏和特异性检测。
Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR-Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.