High-fidelity detection of DNA combining the CRISPR/Cas9 system and hairpin probe
High-fidelity detection of DNA combining the CRISPR/Cas9 system and hairpin probe
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DOI：
10.1016/j.bios.2021.113212
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发表时间：
2021-04-14
影响因子：
12.6
通讯作者：
Li, Jinming
中科院分区：
文献类型：
--
作者：
Wang, Meng;Han, Dongsheng;Li, Jinming
高保真序列测量和单核苷酸变异(SNV)基因分型非常需要能够对DNA进行特异和灵敏检测的方法。CRISPR(簇状规则间隔短重复序列)/CAS系统为检测核酸提供了革命性的工具。然而，目前大多数基于CRISPR/Cas的DNA生物传感平台都受到Cas蛋白固有的脱靶效应的影响，并且需要预扩增过程，这损害了分析的保真度。在这项工作中，使用了CRISPR/Cas9触发的发夹探针介导的生物传感方法(即CHP)来直接读取原始DNA序列，同时有效地中和了脱靶效应并获得了高灵敏度。这项技术可以在阿托摩尔水平上以检出限(LOD)定量DNA靶标，并识别等位基因比例低至0.01%或0.1%的SNV。此外，我们还表明，CHP系统适用于检测血清样本中的突变，而不需要DNA分离步骤。总之，CHP系统是一个灵敏和高保真的平台，有望为DNA序列分析和SNVS基因分型提供强大的工具。
Methods that enable specific and sensitive detection of DNA are greatly required for high-fidelity sequence measurement and single-nucleotide variations (SNVs) genotyping. The CRISPR (clustered regularly interspaced short palindmmic repeats)/Cas systems have provided revolutionary tools for detecting nucleic acids. However, most of the current CRISPR/Cas-based DNA biosensing platforms suffer from inherent off-target effects of Cas proteins and require pre-amplification processes, which compromise the analytical fidelity. In this work, a CRISPR/Cas9-triggered hairpin probe-mediated biosensing method (namely CHP) was used to directly read the original DNA sequences, while effectively neutralizing the off-target effect and achieving high sensitivity. This technique can quantify DNA targets with a limit of detection (LOD) at the attomole level and identify SNVs with allelic fractions as low as 0.01%similar to 0.1%. Moreover, we show that the CHP system is applicable in detecting mutations in serum samples without DNA isolation steps. Collectively, the CHP system is a sensitive and highfidelity platform, which promises a great potential for providing robust tool for DNA sequence analysis and SNVs genotyping.