Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

中文翻译：

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用于独家检测单链 DNA (ssDNA) 的新型抗 CRISPR 辅助 CRISPR 生物传感器

核酸分析在疾病诊断和治疗中发挥着重要作用。 CRISPR技术的发现为核酸检测提供了新颖且通用的方法。然而，最广泛使用的 CRISPR-Cas12a 检测平台缺乏区分单链 DNA (ssDNA) 和双链 DNA (dsDNA) 的能力。为了克服这一限制，我们首先采用抗 CRISPR 蛋白 (AcrVA1) 开发了一种新型 CRISPR 生物传感器来专门检测 ssDNA。在这种传感策略中，AcrVA1 切割 CRISPR 引导 RNA (crRNA)，以抑制 CRISPR-Cas12a 系统的切割活性。只有ssDNA有能力招募切割后的crRNA片段来恢复CRISPR-Cas12生物传感器的检测能力，但dsDNA无法做到这一点。通过测量 CRISPR-Cas12a 生物传感器恢复的切割活性，我们开发的 AcrVA1 辅助 CRISPR 生物传感器能够区分 ssDNA 和 dsDNA，为 ssDNA 的检测提供了一种简单可靠的方法。此外，我们还展示了我们开发的 AcrVA1 辅助 CRISPR 生物传感器来监测解旋酶的酶活性并筛选其抑制剂。