Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the “on–off–on” mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially “blocked” state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the “RESET” effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

中文翻译：

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变构激活剂调节的 CRISPR/Cas12a 系统可实现细胞内内源性和外源性靶标的生物传感和成像

基于CRISPR/Cas12a系统的反式切割活性设计的传感器开辟了生物传感领域的新时代。目前基于 CRISPR/Cas12 的“开-关-开”模式传感器设计主要集中于对激活链 (AS) 进行编程，以响应目标信息间接切换 Cas12a 的反式切割活性。然而，这种设计通常需要额外的辅助探针的帮助，以保持激活链处于最初的“阻断”状态。辅助探针的长度设计和用量需要严格优化，以保证最低的背景和最佳的信噪比。这将不可避免地增加实验的复杂性。为了解决这个问题，我们建议使用“RESET”效应后的AS来直接调节Cas12a酶活性。最初，激活剂链被合理设计为嵌入发夹结构中，以剥夺其激活CRISPR/Cas12a系统的能力。当靶标存在时，靶标介导的链置换引起AS构象变化，发夹结构打开，CRISPR/Cas12a系统重新激活； AS的可切换结构可用于根据目标浓度调节Cas12a的激活程度。由于低背景和稳定性的优势，基于CRISPR/Cas12a的策略不仅可以对活细胞中的内源生物标志物（miR-21）进行成像，还可以对外源病毒入侵细胞的过程进行长期、准确的成像分析。病毒基因组在宿主细胞中的释放和复制是病毒感染细胞不可缺少的标志事件；对它们进行灵敏监测对于揭示病毒感染机制、防御病毒性疾病具有重要意义。