One-Pot Detection of Biomarker Apurinic/Apyrimidinic Endonuclease 1 Based on the Modified-crRNA Regulated Trans-Cleavage Activity of CRISPR/Cas12a

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-08-02 DOI:10.1021/acssynbio.5c00335

Sheng Ding*, Haiyan Li, Jing Li, Dianxiang Lu*, Jin Yang* and Zhuo Tang,

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引用次数: 0

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1), a critical protein in DNA repair, plays indispensable roles in the maintenance of cellular homeostasis, thereby garnering significant attention as a biomarker and therapeutic target for various disorders. Current APE1 sensing methods always require multiple enzymes or complex signal amplification. The high programmability of the CRISPR/Cas12-based signal amplifier provides a new chance for developing biosensors. In this study, we introduce a novel method for the detection of APE1 by leveraging the discovery that modulating the length of modified DNA within CRISPR RNA (crRNA) enables precise control over the trans-cleavage activity of CRISPR/Cas12a. By designing a specific crRNA, the APE1-mediated activity recovery of Cas12a (ARC) was developed for rapid, specific, and one-pot detection of APE1. ARC presented a detection limit of 1.74 × 10^–6 U/μL with high specificity in detecting APE1 in biological samples. Besides, this simple method was feasible for APE1 inhibition assays, highlighting its potential for inhibitor screening and evaluation. Collectively, our findings present an innovative approach for APE1 activity analysis and expand the CRISPR-based non-nucleic acid target sensing toolbox through a novel crRNA design.

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基于修饰的crrna调控CRISPR/Cas12a反式切割活性的生物标志物无尿嘧啶/无嘧啶内切酶1的单锅检测

无尿嘧啶/无嘧啶核酸内切酶1 （APE1）是DNA修复的关键蛋白，在维持细胞稳态中起着不可或缺的作用，因此作为生物标志物和各种疾病的治疗靶点受到了广泛关注。目前的APE1检测方法往往需要多种酶或复杂的信号放大。基于CRISPR/ cas12的信号放大器的高可编程性为生物传感器的开发提供了新的机遇。在这项研究中，我们引入了一种检测APE1的新方法，该方法利用了调节CRISPR RNA （crRNA）内修饰DNA的长度可以精确控制CRISPR/Cas12a的反式切割活性的发现。通过设计特异性的crRNA，开发了APE1介导的Cas12a活性恢复（ARC），用于快速、特异性、一锅检测APE1。ARC检测生物样品中APE1的检出限为1.74 × 10-6 U/μL，特异性高。此外，该方法简便易行，可用于APE1抑制实验，在抑制剂筛选和评价方面具有较大的潜力。总之，我们的研究结果为APE1活性分析提供了一种创新的方法，并通过一种新的crRNA设计扩展了基于crispr的非核酸靶标传感工具箱。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.