Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA

被引：8

作者：

He, Wen ^{[1
]}

Li, Xinyu ^{[1
]}

Li, Xinmin ^{[2
]}

Guo, Minghui ^{[1
]}

Zhang, Mengxuan ^{[1
]}

Hu, Ruiwei ^{[1
]}

Li, Menghan ^{[1
]}

Ding, Shijia ^{[1
]}

Yan, Yurong ^{[1
]}

机构：

[1] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China

[2] Chongqing Hosp Tradit Chinese Med, Chongqing Key Lab Sichuan Chongqing Coconstruct Di, Chongqing 400021, Peoples R China

来源：

ANALYTICA CHIMICA ACTA | 2024年 / 1303卷

基金：

中国国家自然科学基金;

关键词：

CRISPR/Cas12a; Chimeric DNA-RNA hybrid activator; Split activator; RNA detection; TRANS-CLEAVAGE; CRISPR-CAS12A;

D O I：

10.1016/j.aca.2024.342477

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

引用

页数：8