State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha 410082, P. R. China.
Institute of Pathogen Biology and Immunology of College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China.
Sci Adv. 2021 Jan 27;7(5). doi: 10.1126/sciadv.abc7802. Print 2021 Jan.
Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.
具有精确可控动态和功能的人工核酸电路在生物传感中显示出巨大的应用前景，但由于无法直接处理基因组 DNA 和中等灵敏度，其在分子诊断中的应用仍然受到限制。为了解决这一限制，我们提出了一种基于 CRISPR-Cas 的催化核酸电路，即 CRISPR-Cas 仅扩增网络 (CONAN)，用于等温扩增检测基因组 DNA。通过整合 Cas12a 的严格靶标识别、解旋酶活性和转切割活性，设计了一个 Cas12a 自动催化驱动的人工反应网络，在 CONAN 中构建具有指数动力学的正反馈电路。因此，CONAN 实现了单酶、单步、实时检测基因组 DNA，具有亚皮摩尔灵敏度。此外，CONAN 提高了 Cas12a 的固有单碱基特异性，并能够有效检测临床样本中的乙型肝炎病毒感染和人类膀胱癌相关单核苷酸突变，突出了其作为疾病诊断工具的潜力。
