LbuCas13a directly targets DNA and elicits strong trans-cleavage activity

IF 26.8 1区医学 Q1 ENGINEERING, BIOMEDICAL

Nature Biomedical Engineering Pub Date : 2025-06-20 DOI:10.1038/s41551-025-01424-6

Xiaolong Wu, Siyuan Luo, Chuanghao Guo, Yi Zhao, Jialing Zhong, Ronghuan Hu, Xinyao Yang, Conghui Liu, Qianling Zhang, Songkuan Zhuang, Yong Chen, Yizhen Liu, Xueji Zhang

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Abstract

Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of Leptotrichia buccalis Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and protospacer adjacent motif sequences, coupled with robust trans-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA–DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl⁻¹). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics.

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LbuCas13a直接靶向DNA并引发强烈的反式切割活性

传统上被认为是一种RNA特异性核酸酶，Cas13a主要用于RNA检测。我们发现钩毛细毛菌Cas13a （LbuCas13a）能够直接靶向DNA，而不受原间隔子侧翼序列和原间隔子相邻基序序列的限制，并且具有强大的反式切割活性。与传统认识相反，LbuCas13a不会降解DNA靶标。我们的研究表明，与RNA相比，LbuCas13a对DNA的单核苷酸特异性增强。这种增强的特异性归因于CRISPR RNA （crRNA）对DNA的亲和力较低，从而提高了crRNA - DNA的结合能屏障。我们推出了一种分子诊断平台，称为LbuCas13a的superior universal rapid enhanced specificity test (SUREST)，用于高分辨率基因分型。SUREST能够在0.3 aM （0.18 cpsµl−1）内检测CYP2C19 （rs4986893）的DNA浓度。我们还将SUREST应用于人类基因分型场景，表明SUREST在广泛的突变和序列背景下表现良好。SUREST代表了实时核酸检测的进步，使其成为临床诊断中病原体鉴定和突变分析的有用工具。

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

Nature Biomedical Engineering Medicine-Medicine (miscellaneous)

CiteScore

45.30

自引率

1.10%

发文量

138

期刊介绍： Nature Biomedical Engineering is an online-only monthly journal that was launched in January 2017. It aims to publish original research, reviews, and commentary focusing on applied biomedicine and health technology. The journal targets a diverse audience, including life scientists who are involved in developing experimental or computational systems and methods to enhance our understanding of human physiology. It also covers biomedical researchers and engineers who are engaged in designing or optimizing therapies, assays, devices, or procedures for diagnosing or treating diseases. Additionally, clinicians, who make use of research outputs to evaluate patient health or administer therapy in various clinical settings and healthcare contexts, are also part of the target audience.