CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.

IF 13.1 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research Pub Date : 2025-09-05 DOI:10.1093/nar/gkaf893

Qingyuan Jiang,Shuqi Jin,Zhichao Qin,Junqi Zhang,Ruyi He,Zhuo Chen,Bin Qiao,Jie Qiao,Yi Liu

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引用次数: 0

Abstract

The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.

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CRISPR/Cas12a DTR系统：一种拓扑引导的Cas12a检测，用于特异性双重检测RNA和DNA靶标。

CRISPR/Cas12a技术彻底改变了分子诊断。然而，现有的Cas12a系统依赖于连续的靶标DNA激活，这限制了它们只能进行单靶标检测。在这项研究中，我们开发了一种新的拓扑引导Cas12a系统，即双靶标响应（DTR）系统，能够被不连续的双RNA/DNA靶标激活。DTR系统采用两个分裂的CRISPR RNA （crRNA）片段和两个Cas12a蛋白，在识别两种核酸激活剂后协同重建。我们证明了DTR系统在单次读数中特异性检测双核酸底物的能力，对RNA的检测限为78 fM，对单核苷酸变异的特异性非常高。此外，我们成功地将DTR系统应用于临床样本，能够同时检测两种口腔鳞状细胞癌相关的microrna (miR-155和miR-let-7a)，从而区分健康个体和患者。本工作建立了一个高效的基于cas12的平台，对RNA和DNA靶点进行灵敏、同步、鉴别检测，增强了Cas12a在分析检测和临床诊断中的通用性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Nucleic Acids Research 生物-生化与分子生物学

CiteScore

27.10

自引率

4.70%

发文量

1057

审稿时长

2 months

期刊介绍： Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.