中国科学院长春应用化学研究所李冰凌团队报道了，基于模块化和可定制的CRISPR/Cas辅助纳米孔操作连接（CANON）的精确分子传感。相关研究成果发表在2025年1月13日出版的《德国应用化学》。

固态纳米孔是一种有前景的单分子检测技术，但在很大程度上受到对小尺寸靶标分辨率相对较低和信号探针设计繁琐的限制。

该文中，研究人员建立了一个通用的CRISPR/Cas辅助纳米孔操作连接（CANON），它可以通过“信号开启”模式将不同的靶向来源/物种准确地转导到不同的DNA结构探针中。靶识别激活Cas12a/crRNA系统的切割活性，然后完全消化引发剂的阻断剂。然后，未堵塞的引发剂引发下游DNA组装反应，并产生易于纳米孔检测的大尺寸结构。Cas12a/crRNA与DNA组装的这种整合在输入靶标、输出DNA结构和最终的纳米孔信号之间建立了精确的对应关系。

研究人员展示了dsDNA、长RNA（即流感病毒基因）、短microRNA（即let-7d）和非核酸（即Pb²⁺）作为输入范式。各种结构组装反应，如杂交链式反应（HCR）、G-HCR和双链聚合策略（DPS），被用作纳米孔信号的输出。通过将FluA和FluB基因分别转移到HCR和G-HCR中，也验证了同时检测。因此，CANON是一个模块化的传感平台，具有高精度、高分辨率和高通用性等多重优势，可以轻松定制到各种应用场景中。

附：英文原文

Title: Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON)

Author: Huaning Wang, Rujian Zhao, Bing Zhang, Yao Xiao, Chunmiao Yu, Yesheng Wang, Chunxu Yu, Yidan Tang, Yanru Li, Baiyang Lu, Bingling Li

Issue&Volume: 2025-01-13

Abstract: Solid-state nanopore is a promising single molecular detection technique, but is largely limited by relatively low resolution to small-size targets and laborious design of signaling probes. Here we establish a universal, CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON), which can accurately transduce different targeting sources/species into different DNA structural probes via a “Signal-ON” mode. Target recognition activates the cleavage activity of a Cas12a/crRNA system and then completely digest the blocker of an initiator. The unblocked initiator then triggers downstream DNA assembly reaction and generate a large-size structure easy for nanopore detection. Such integration of Cas12a/crRNA with DNA assembly establishes an accurate correspondence among the input targets, output DNA structures, and the ultimate nanopore signals. We demonstrated dsDNA, long RNA (i.e., Flu virus gene), short microRNA (i.e., let-7d) and non-nucleic acids (i.e., Pb2+) as input paradigms. Various structural assembly reactions, such as hybridization chain reaction (HCR), G-HCR and duplex polymerization strategy (DPS), are adapted as outputs for nanopore signaling. Simultaneous assay is also verified via transferring FluA and FluB genes into HCR and G-HCR, respectively. CANON is thus a modular sensing platform holding multiple advantages such as high accuracy, high resolution and high universality, which can be easily customized into various application scenes.

DOI: 10.1002/anie.202423473

Source: https://onlinelibrary.wiley.com/doi/10.1002/anie.202423473