Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA

IF 5.7 2区化学 Q1 CHEMISTRY, ANALYTICAL

Analytica Chimica Acta Pub Date : 2025-05-17 DOI:10.1016/j.aca.2025.344204

Wenjing Ren, Mingzhi Li, Xinyue Liu, Weixin You, Qianqian You, Boan Li, Huiming Ye, Rui Zhang

{"title":"Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA","authors":"Wenjing Ren, Mingzhi Li, Xinyue Liu, Weixin You, Qianqian You, Boan Li, Huiming Ye, Rui Zhang","doi":"10.1016/j.aca.2025.344204","DOIUrl":null,"url":null,"abstract":"<h3>Background</h3>The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.<h3>Results</h3>We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an \"AND\" logic gate detection platform that processes multiple inputs within 40 minutes. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.<h3>Significance</h3>SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.","PeriodicalId":240,"journal":{"name":"Analytica Chimica Acta","volume":"30 1","pages":""},"PeriodicalIF":5.7000,"publicationDate":"2025-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytica Chimica Acta","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1016/j.aca.2025.344204","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}

引用次数: 0

Abstract

Background

The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.

Results

We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an "AND" logic gate detection platform that processes multiple inputs within 40 minutes. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.

Significance

SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.

Abstract Image

查看原文本刊更多论文

利用含有间隔分裂crRNA的CRISPR-Cas12a系统特异性检测DNA和RNA

CRISPR/Cas12a系统由于其双重顺式和反式切割活性而成为一种多功能的分子诊断工具。然而，两个关键的限制阻碍了它的广泛应用：对DNA靶点单碱基错配的高耐受性和对DNA激活剂的严格依赖。为了解决这些挑战，我们假设crRNA的结构重组可以增强特异性和功能的多功能性。本研究旨在开发一种改进的Cas12a系统，能够以更高的单碱基分辨率检测DNA和RNA靶标，从而扩大其在分子诊断和临床亚分类中的应用。我们通过在间隔区引入一个分裂位点来设计分裂crrna，创建了一个间隔区分裂crrna激活的Cas12a系统（SPCas12a）。该系统表现出三个关键优势：首先，与传统的全尺寸crRNA系统相比，SPCas12a在区分单碱基突变方面表现出显著增强的特异性。其次，它绕过了DNA激活剂的要求，无需逆转录即可直接检测miRNA靶点。此外，AlphaFold Server预测结构建模分析显示，SPCas12a选择的分裂位点使Cas12a复合体具有开放的结构域，有利于Cas12a功能的稳定。第三，与等温放大集成，构建了一个“与”逻辑门检测平台，可在40分钟内处理多个输入。作为概念验证，SPCas12a通过分析不同细胞系中的miRNA-210和miRNA-21生物标志物成功区分三阴性乳腺癌（TNBC）亚型细胞系。espcas12a通过在单一平台上统一高特异性DNA突变检测和直接RNA传感，克服了当前CRISPR诊断的基本局限性。分裂- crrna设计原则提供了一种普遍适用的策略来增强CRISPR-Cas系统，可立即应用于精确肿瘤学和传染病分层，在这些领域，基础水平的区分和多靶点检测是至关重要的。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Analytica Chimica Acta 化学-分析化学

CiteScore

10.40

自引率

6.50%

发文量

1081

审稿时长

38 days

期刊介绍： Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.