CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2024-11-20 DOI:10.1021/acssynbio.4c00265

Koji Asano, Kazuto Yoshimi, Kohei Takeshita, Satomi Mitsuhashi, Yuta Kochi, Rika Hirano, Zong Tingyu, Saeko Ishida, Tomoji Mashimo

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引用次数: 0

Abstract

Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.

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用于定量和快速诊断肌营养不良 1 型重复扩增症的 CRISPR 诊断技术。

以肌萎缩症 1 型（DM1）为例，由于重复长度的可变性和复杂性，重复扩增疾病给诊断量化带来了挑战。包括 PCR 和 Southern 印迹在内的传统诊断方法在灵敏度和特异性方面存在局限性，因此有必要开发创新方法来进行精确、快速的诊断。在此，我们介绍一种基于 CRISPR 的诊断方法 REPLICA（通过 Cas3 对遗传病进行重复定位），用于 DM1 的定量和快速诊断。该方法使用体外组装的CRISPR-Cas3，在量化CTG重复扩增长度方面表现出卓越的灵敏度和特异性，并与疾病的严重程度相关。我们还利用患者基因组验证了 CRISPR 诊断在定量诊断 DM1 方面的稳健性和准确性。此外，我们还优化了基于 REPLICA 的检测方法，以便使用侧流试纸进行护理点检测，从而促进快速筛查和检测。总之，基于 REPLICA 的 CRISPR 诊断可精确、快速地检测重复扩增疾病，有望实现个性化治疗策略。

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

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.