<br>使用自激活 CRISPR-Cas12a 生物传感器的 DNAzyme 触发平衡转移可实现核酸的一锅法诊断,Analytical Chemistry

当前位置： X-MOL 学术 › Anal. Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

使用自激活 CRISPR-Cas12a 生物传感器的 DNAzyme 触发平衡转移可实现核酸的一锅法诊断
Analytical Chemistry ( IF 6.7 ) Pub Date : 2025-01-31 , DOI: 10.1021/acs.analchem.4c06066
Di Huang _{1,

2} , Yichen He _{1,

2} , Chutian Xu ₃ , Peijie Shen _{1,

2} , Min Li ₄ , Mengjun Fang ₅ , Zhinan Xu _{1,

2,

6} , Xiangming Fang ₄

Affiliation

将重组酶聚合酶扩增（RPA）与 CRISPR-Cas12a 相结合，在简化和改进核酸诊断程序方面具有巨大潜力。然而，当前的策略面临局限性，例如复杂性、效率降低和 Cas12a 活性的潜在妥协。作为回应，我们开发了一种 DNAzyme 触发的 equilibrium 转移，其中 self 激活的 CRISPR-Cas12a biosensor （DESCRIBER）用于整合核酸检测。该平台具有不同的平衡点，以最大限度地减少 RPA 和 Cas12a 在一个罐中的干扰，并最大限度地提高它们在不同阶段的活性。最初，反应集中在 RPA 上，而 Cas12a 被环状 crRNA （C-crRNA）沉默。然后，在 RPA 过程中产生激活剂 DNAzyme，该激活剂使 C-crRNA 线性化以激活 Cas12a 并将平衡转移到信号读数。同时，活化的 Cas12a 可以进一步线性化 C-crRNA 以促进自我激活并加速平衡转移。根据这一原理，在 1 小时内实现了低至 500 CPs/mL 的 HIV-1 基因组的高灵敏度检测，同时保持了检测常见亚型的普遍性和对机会性感染性病原体的特异性。与 qRT-PCR 相比，它在检测 35 个加标样品时也表现出良好的准确性。总体而言，我们相信拟议的策略将增强现有的 CRISPR 系统，以促进它们在临床诊断中的实际应用。

"点击查看英文标题和摘要"

DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids

Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer with a self-activated CRISPR-Cas12a biosensor (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.

更新日期：2025-01-31

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南