Zhu Fengxi, Zhao Qiang
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2025 Jun 17;97(23):12215-12222. doi: 10.1021/acs.analchem.5c00937. Epub 2025 Jun 5.
Discovery of CRISPR/Cas12a has revolutionized broad fields, including gene editing, molecular diagnosis, and biosensing. Flexible regulation of Cas12a activity is important for diverse CRISPR/Cas12a applications, especially for biosensing, but it still faces limitations and challenges. We find gold nanoparticles (AuNPs) modified with a single-stranded DNA activator create a huge steric barrier that strongly locks activators in a one-to-many manner and inhibits Cas12a activity. This finding offers a new way to modulate the activity of Cas12a for applications, such as designing versatile biosensors. We report a spherical nucleic acid (SNA)-modulating CRISPR/Cas12a (SNA-Cas) platform using SNAs as signal translators for target sensing. A stimuli-responsive SNA was constructed by modifying AuNPs with a DNA activator containing a specific trigger element, and the target triggers specific reactions (e.g., thiol-exchange chemical reaction and RNA-cleaving by DNAzymes) to release activators into solution. A free activator initiates -cleavage activity of CRISPR/Cas12a, scissoring fluorescent DNA reporters to produce amplified signals. To show proof of concept, we demonstrate SNA-Cas to sensitively detect diverse non-nucleic acid targets, including biological thiol cysteine, heavy metal Pb, and biomarkers of O-methylguanine-DNA-methyltransferases (MGMT) and fat mass and obesity-related protein (FTO) demethylases. This work opens one door for SNA modulating CRISPR/Cas activity and shows great potential in designing versatile biosensors for detecting diverse targets.
CRISPR/Cas12a的发现给包括基因编辑、分子诊断和生物传感在内的广泛领域带来了变革。灵活调控Cas12a活性对于多种CRISPR/Cas12a应用至关重要，尤其是对于生物传感，但它仍然面临局限性和挑战。我们发现用单链DNA激活剂修饰的金纳米颗粒（AuNP）会形成巨大的空间位垒，以一对一的方式强烈锁定激活剂并抑制Cas12a活性。这一发现为调节Cas12a活性以用于诸如设计通用生物传感器等应用提供了一种新方法。我们报道了一种球形核酸（SNA）调节的CRISPR/Cas12a（SNA-Cas）平台，该平台使用SNA作为用于目标传感的信号转导器。通过用含有特定触发元件的DNA激活剂修饰AuNP构建了一种刺激响应性SNA，目标触发特定反应（例如硫醇交换化学反应和DNA酶切割RNA）以将激活剂释放到溶液中。游离的激活剂启动CRISPR/Cas12a的切割活性，切割荧光DNA报告分子以产生放大信号。为了证明概念，我们展示了SNA-Cas能够灵敏地检测多种非核酸目标，包括生物硫醇半胱氨酸、重金属铅以及O-甲基鸟嘌呤-DNA甲基转移酶（MGMT）和脂肪量与肥胖相关蛋白（FTO）去甲基化酶的生物标志物。这项工作为SNA调节CRISPR/Cas活性打开了一扇门，并在设计用于检测多种目标的通用生物传感器方面显示出巨大潜力。
