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

作者全名：He, Wen; Li, Xinyu; Li, Xinmin; Guo, Minghui; Zhang, Mengxuan; Hu, Ruiwei; Li, Menghan; Ding, Shijia; Yan, Yurong

作者地址：[He, Wen; Li, Xinyu; Guo, Minghui; Zhang, Mengxuan; Hu, Ruiwei; Li, Menghan; Ding, Shijia; Yan, Yurong] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Li, Xinmin] Chongqing Hosp Tradit Chinese Med, Chongqing Key Lab Sichuan Chongqing Coconstruct Di, Chongqing 400021, Peoples R China

通信作者：Yan, YR (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China.

来源：ANALYTICA CHIMICA ACTA

ESI学科分类：CHEMISTRY

WOS号：WOS:001215524000001

JCR分区：Q1

影响因子：5.7

年份：2024

卷号：1303

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：CRISPR/Cas12a; Chimeric DNA-RNA hybrid activator; Split activator; RNA detection

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.

基金机构：National Natural Science Foundation of China [81873980, 82102505]; Natural Science Foundation Project of Chongqing [CSTB2023NSCQ- MSX0915]

基金资助正文：This work was supported by financial support from the National Natural Science Foundation of China (81873980, 82102505) and the Natural Science Foundation Project of Chongqing (CSTB2023NSCQ- MSX0915) .