Trans-cleavage activity of Cas12a effectors can be unleashed by both double-stranded DNA and single-stranded RNA targeting in absence of PAM

IF 8.5 1区化学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

International Journal of Biological Macromolecules Pub Date : 2025-04-11 DOI:10.1016/j.ijbiomac.2025.142992

Guohui Xiao , Hongyu Shi , Meixia Liu , Min Huang , Siqi Li , Xuefeng Zhou , Hengfei Li , Guoliang Zhang

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引用次数: 0

Abstract

CRISPR-Cas12a is a powerful tool in nucleic acid detection, but the relationship between its trans-cleavage activity and protospacer adjacent motif (PAM) sequences remains incompletely understood. In this study, we synthesized diverse PAM-sequence substrates and conducted systematic cis-cleavage and trans-cleavage experiments with three Cas12a orthologs. We found that double-stranded DNA (dsDNA) can activate Cas12a's trans-cleavage activity even without PAM and this activation occurring independently of cis-cleavage. Notably, our results also revealed that single-stranded RNA (ssRNA) can directly initiate the trans-cleavage activity of Cas12a.We also experimentally validated the feasibility of CRISPR-Cas12a in detecting target dsDNA lacking PAM sequences, including identifying mutated sites in clinical samples. Structural prediction using AlphaFold 3 revealed the potential mechanism of Cas12a's PAM-independent trans-cleavage. Our research expands the understanding of Cas12a's trans-cleavage mechanism and demonstrates its potential for nucleic acid detection beyond PAM-dependent targets. This discovery broadens the application scope of Cas12a, providing new opportunities for developing highly sensitive and versatile diagnostic platforms.

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在没有PAM的情况下，Cas12a效应物的反式切割活性可以通过双链DNA和单链RNA靶向来释放

CRISPR-Cas12a是一种强大的核酸检测工具，但其反式切割活性与原间隔器邻近基序（protospacer邻基序，PAM）序列之间的关系尚不完全清楚。在本研究中，我们合成了多种pam序列底物，并对三个Cas12a同源物进行了系统的顺式和反式切割实验。我们发现，即使没有PAM，双链DNA （dsDNA）也可以激活Cas12a的反式切割活性，并且这种激活独立于顺式切割发生。值得注意的是，我们的研究结果还揭示了单链RNA （ssRNA）可以直接启动Cas12a的反式切割活性。我们还通过实验验证了CRISPR-Cas12a检测缺乏PAM序列的靶dsDNA的可行性，包括在临床样本中识别突变位点。利用AlphaFold 3进行结构预测，揭示了Cas12a不依赖pam的反式切割的潜在机制。我们的研究扩大了对Cas12a反式切割机制的理解，并证明了它在pam依赖靶标之外的核酸检测方面的潜力。这一发现拓宽了Cas12a的应用范围，为开发高灵敏度和多用途的诊断平台提供了新的机会。

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

International Journal of Biological Macromolecules 生物-生化与分子生物学

CiteScore

13.70

自引率

9.80%

发文量

2728

审稿时长

64 days

期刊介绍： The International Journal of Biological Macromolecules is a well-established international journal dedicated to research on the chemical and biological aspects of natural macromolecules. Focusing on proteins, macromolecular carbohydrates, glycoproteins, proteoglycans, lignins, biological poly-acids, and nucleic acids, the journal presents the latest findings in molecular structure, properties, biological activities, interactions, modifications, and functional properties. Papers must offer new and novel insights, encompassing related model systems, structural conformational studies, theoretical developments, and analytical techniques. Each paper is required to primarily focus on at least one named biological macromolecule, reflected in the title, abstract, and text.