Rapid and Sensitive Detection of USP26 Cluster Variants Based on CRISPR/Cas12a

IF 1.8 2区医学 Q2 GENETICS & HEREDITY

Human Mutation Pub Date : 2026-10-13 DOI:10.1155/humu/5152235

Ning Li, Yongkun Huo, Yanan Zhao, Chao Liu, Zhe Zhang, Kaizhao Chen, Longjun Chen, Xiaonan Liu, Ziyang Lin, Ling Sun, Hui Jiang, Xinjie Wang, Wei Li

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引用次数: 0

Abstract

USP26 gene variants have been reported to be associated with male infertility, such as non-obstructive azoospermia (NOA), oligozoospermia, and asthenoteratozoospermia, and may be associated with altered sperm sex chromosome segregation patterns. Among the reported variants, USP26 cluster variants (c.370_371insACA/c.494 T > C/c.1423C > T) represent a frequently reported variant pattern in certain populations. Rapid and convenient detection of USP26 cluster variants may serve as a powerful tool for male infertility-associated cohorts. Here, we develop a CRISPR/Cas12a-based assay to rapid detection USP26 variants in human samples. Candidate CRISPR RNAs (crRNAs) and RPA primers were screened and optimized, followed by incorporation of restriction enzymes to reduce wild-type–derived amplification interference. Using controlled artificial DNA mixtures, the assay detected mutant-template fractions down to 5% under the tested conditions. The complete workflow could be performed within 60 min using basic laboratory equipment. The results of the CRISPR/Cas12a system are consistent with Sanger sequencing in 91 male infertility research samples. Overall, our study establishes a rapid molecular detection method for detecting USP26 cluster variants and provides an alternative approach for genetic studies of male infertility-associated cohorts.

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基于CRISPR/Cas12a的USP26聚类变异快速灵敏检测

据报道，USP26基因变异与男性不育有关，如非阻塞性无精子症（NOA）、少精症和弱异精症，并可能与精子性染色体分离模式改变有关。在报告的变体中，USP26集群变体（C . 370_371insaca / C .494 T > C/ C . 1423c > T）代表了某些人群中经常报道的变体模式。快速方便地检测USP26聚类变异可作为男性不育相关队列的有力工具。在这里，我们开发了一种基于CRISPR/ cas12的检测方法来快速检测人类样本中的USP26变体。筛选和优化候选CRISPR rna （crrna）和RPA引物，随后加入限制性内切酶以减少野生型衍生的扩增干扰。使用受控的人工DNA混合物，在测试条件下检测到突变模板分数低至5%。使用基本的实验室设备，完整的工作流程可在60分钟内完成。CRISPR/Cas12a系统的结果与91个男性不育症研究样本的Sanger测序结果一致。总的来说，我们的研究建立了一种快速的分子检测方法来检测USP26簇变异体，并为男性不育相关队列的遗传研究提供了一种替代方法。

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

Human Mutation 医学-遗传学

CiteScore

8.40

自引率

5.10%

发文量

190

审稿时长

2 months

期刊介绍： Human Mutation is a peer-reviewed journal that offers publication of original Research Articles, Methods, Mutation Updates, Reviews, Database Articles, Rapid Communications, and Letters on broad aspects of mutation research in humans. Reports of novel DNA variations and their phenotypic consequences, reports of SNPs demonstrated as valuable for genomic analysis, descriptions of new molecular detection methods, and novel approaches to clinical diagnosis are welcomed. Novel reports of gene organization at the genomic level, reported in the context of mutation investigation, may be considered. The journal provides a unique forum for the exchange of ideas, methods, and applications of interest to molecular, human, and medical geneticists in academic, industrial, and clinical research settings worldwide.