CRISPR/Cas系统在分子诊断领域的应用研究进展

doi:10.12211/2096-8280.2025-042

摘要/Abstract

摘要：

CRISPR/Cas系统因其高特异性、可编程性和便捷性，已成为分子诊断领域的重要工具。本文综述了CRISPR/Cas系统的技术原理、诊断平台优化及其在精准医学中的应用进展。首先，概述了CRISPR/Cas系统的作用机制与分类，并重点讨论了CRISPR诊断技术的创新优化策略，包括基于核酸预扩增（如SHERLOCK（specific high-sensitivity enzymatic reporter unlocking）、DETECTR（DNA endonuclease targeted CRISPR trans reporter））和免扩增的检测方法。其次，探讨了CRISPR/Cas技术在感染性疾病（病原体筛查、耐药性检测）、肿瘤分子分型（癌症早筛、遗传变异分析）及非核酸标志物检测中的临床应用。最后，本文展望了该技术的未来发展方向，包括微型化设备开发、高通量智能化诊断体系构等，并分析了其在临床转化中面临的关键挑战（如灵敏度标准化、成本控制等）。通过总结目前研究，本文旨在为CRISPR/Cas技术在分子诊断领域的进一步优化和医学应用提供理论参考。

关键词: CRISPR/Cas系统, 即时检测, 临床转化应用, 微型化设备, 人工智能辅助分子诊断

Abstract:

The CRISPR/Cas system has emerged as a pivotal tool in molecular diagnostics due to its high specificity, programmability, and ease of use. This review provides a comprehensive overview of the mechanistic principles, diagnostic platform optimization, and applications of CRISPR/Cas systems in precision medicine. We begin with an outline of the classification and molecular mechanisms of CRISPR/Cas systems, highlighting the functional differences among Type I-VI systems. Our focus then shifts to innovative diagnostic strategies, including nucleic acid pre-amplification methods (e.g., SHERLOCK, DETECTR) and amplification-free approaches. These strategies have significantly enhanced the sensitivity and specificity of molecular diagnostics, making them more efficient and reliable than conventional techniques. The review then explores the broad clinical applications of CRISPR/Cas technology in infectious diseases, such as pathogen screening and drug-resistance detection, demonstrating remarkable utility in rapidly identifying pathogens and their resistance profiles. In the field of cancer research, the technology has shown great potential in early screening and genetic variant analysis, which are crucial for developing personalized treatment strategies. Moreover, CRISPR/Cas systems are expanding their diagnostic capabilities to include non-nucleic acid biomarker detection, further solidifying their position as versatile tools in clinical diagnostics. The review also addresses future directions in the field, such as the development of miniaturized devices and high-throughput intelligent diagnostic systems. These advancements are expected to enhance portability, accessibility, and efficiency, enabling rapid point-of-care testing in diverse settings, including resource-limited environments. The integration of CRISPR technology with microfluidics and portable detection devices is particularly promising for enabling rapid and accurate diagnostics at the bedside or in the field. As research continues to evolve, the ongoing refinement of CRISPR/Cas systems is anticipated to significantly improve the accuracy, speed, and accessibility of molecular diagnostics. This progress is expected to lead to better clinical outcomes and more effective public health responses. However, several challenges in clinical translation must be addressed, such as standardization of sensitivity and cost reduction. This review aims to provide a theoretical foundation for advancing CRISPR-based diagnostics in biomedical research and to guide the future development of CRISPR/Cas technologies in molecular diagnostics, particularly in overcoming the limitations of traditional diagnostic methods.

Key words: CRISPR/Cas system, point-of-care testing, clinical translational applications, miniaturized devices, artificial intelligence assisted molecular diagnosis

中图分类号:

Q816

王珂, 陈文慧, 雷春阳, 聂舟. CRISPR/Cas系统在分子诊断领域的应用研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-042.

WANG Ke, CHEN Wenhui, LEI Chunyang, NIE Zhou. Advances in the Application of CRISPR/Cas Systems in Molecular Diagnostics[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-042.

图/表 10

表1 CRISPR效应蛋白的相关特征

Table 1 The relation of CRISPR/Cas system effector proteins

Cas蛋白	大类	型	靶标	反式切割活性/底物	PAM/PFS序列
Cas9^{[2, 3]}	Class II系统	Type II	dsDNA	无	3′ GC-rich PAM
Cas7-11^[4-6] （多亚基复合物）	Class I系统	Type III	ssRNA	无	---
Cas12a (Cpf1)^[7]	Class II系统	Type V	dsDNA/ssDNA	有/ssDNA	5′ AT-rich PAM
Cas12b^[8]	Class II系统	Type V	dsDNA/ssDNA	有/ssDNA	5′ AT-rich PAM
Cas12f (Cas14)^[9-13]	Class II系统	Type V	dsDNA/ssDNA	有/ssDNA	5′ T-rich PAM
Cas13^[14]	Class II系统	Type VI	ssRNA	有/ssRNA	3′ non-G-PFS
Cas3蛋白（多亚基复合物）^[15]	Class I系统	Type I	dsDNA	有/ssDNA	AAG

图1 用于生物传感和生物成像的CRISPR/Cas系统的原理图

Fig. 1 Schematic diagram and principles of CRISPR/Cas systems for biosensing and bioimaging

图2 基于核酸预扩增的CRISPR/Cas分子诊断技术原理图

Fig. 2 Schematic of CRISPR/Cas molecular diagnostic technology based on nucleic acid pre amplification

图3 无核酸预扩增的免扩增CRSIPR技术策略

Fig. 3 Amplification-free CRISPR strategy without nucleic acid pre-amplification

图4 CRISPR/Cas系统用于感染性疾病精准诊断的检测方法原理图

Fig. 4 Schematic diagram of detection methods using the CRISPR/Cas system for precise diagnosis of infectious diseases

图5 CRISPR/Cas系统在癌症生物标志物检测中的应用

Fig. 5 Application of CRISPR/Cas System in Cancer Biomarker Detection

图6 CRISPR/Cas系统用于检测蛋白的方法

Fig. 6 Method for detecting proteins based on CRISPR/Cas system

图7 利用CRISPR/Cas系统检测抗体/代谢物的方法

Fig. 7 Antibody/metabolite detection technologies utilizing the CRISPR/Cas system

图8 CRISPR/Cas系统在分子诊断中结合微型化与集成化技术

Fig. 8 The CRISPR/Cas system combines miniaturization and integration techniques in molecular diagnostics

图9 CRISPR/Cas系统在分子诊断中的挑战

Fig. 9 The Challenges of CRISPR/Cas system in molecular diagnosis

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