使用单一 Cas 蛋白进行基于 CRISPR 的多重核酸检测,Analytical Chemistry

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使用单一 Cas 蛋白进行基于 CRISPR 的多重核酸检测
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-10-26 , DOI: 10.1021/acs.analchem.3c01861
Jinyu Fu ₁ , Linghao Zhang ₁ , Yanlin Long ₁ , Zihe Liu ₁ , Geng Meng ₂ , Huimin Zhao ₃ , Xin Su ₁ , Shuobo Shi ₁

Affiliation

由于其简单、快速和灵敏，基于 CRISPR 的核酸检测已越来越多地用于分子诊断。然而，其主要局限性之一是缺乏多重检测能力，因为检测依赖于Cas蛋白的反式切割活性，这需要使用多个正交Cas蛋白进行多重检测。在此，我们报告了使用单一 Cas 蛋白 (Cas12a) 开发具有单核苷酸分辨率的基于 CRISPR 的多重核酸检测系统。这种方法被称为CRISPR-TMSD，集成了立足点介导的链置换（TMSD）反应，并且由于使用了靶标特异性报告基因，因此使用单个Cas蛋白即可实现Cas蛋白的顺式切割活性和多重检测。使用一套计算模拟工具包来设计 TMSD 报告基因，从而可以对目标序列进行高度灵敏和特异性的识别。与重组酶聚合酶扩增（RPA）结合，检测限可低至1 copy/μL。作为概念证明，CRISPR-TMSD 随后被用于以单核苷酸分辨率检测致癌基因和 SARS-CoV-2 RNA。这项工作代表了一种设计基于 CRISPR 的诊断系统的新概念策略，并且具有扩展基于 CRISPR 的诊断应用的巨大潜力。

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Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein

Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/μL. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.

更新日期：2023-10-26

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