Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing,Analytical Chemistry

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Switching the Activity of CRISPR/Cas12a Using an Allosteric Inhibitory Aptamer for Biosensing
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-11-03 , DOI: 10.1021/acs.analchem.2c04315
Peipei Qin ₁ , Pinru Chen ₁ , Nan Deng ₁ , Liu Tan ₁ , Bin-Cheng Yin _{1,

2} , Bang-Ce Ye _{1,

2}

Affiliation

The current CRISPR/Cas12a-based diagnostic techniques focus on designing the crRNA or substrate DNA elements to indirectly switch the trans-cleavage activity of Cas12a responsive to target information. Here, we propose the use of an allosteric DNA probe to directly regulate the trans-cleavage activity of Cas12a and present a method for sensing different types of analytes. An allosteric inhibitor probe is rationally designed to couple the target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system and enables binding to a specific target to induce the change of conformation, which leads to the loss of its inhibitory function on Cas12a. As a result, the structure-switchable probe can regulate the degree of activity of Cas12a depending on the dose of target. Scalability of our strategy can be achieved by simply replacing the loop domain with different target recognition sequences. The proposed method was validated by detecting adenosine triphosphate and let-7a, giving the detection limits of 490 nM and 26 pM, respectively, and showing an excellent specificity. We believe that this work exploits a viable approach to use the inhibitory aptamer of Cas12a as a regulatory element for biosensing purposes, enriching the arsenal of CRISPR/Cas12a-based methods for molecular diagnostics and spurring further development and application of aptamers of the CRISPR/Cas system.

中文翻译：

使用用于生物传感的变构抑制适配体切换 CRISPR/Cas12a 的活性

目前基于 CRISPR/Cas12a 的诊断技术侧重于设计 crRNA 或底物 DNA 元件，以间接切换 Cas12a 的反式切割活性以响应目标信息。在这里，我们建议使用变构 DNA 探针直接调节 Cas12a 的反式切割活性，并提出一种检测不同类型分析物的方法。合理设计变构抑制剂探针，将靶标识别序列与CRISPR/Cas12a系统的抑制适配体偶联，使其与特定靶标结合，诱导构象发生变化，从而导致其对Cas12a的抑制功能丧失。因此，结构可切换探针可以根据靶标剂量调节 Cas12a 的活性程度。我们的策略的可扩展性可以通过简单地用不同的目标识别序列替换循环域来实现。通过检测三磷酸腺苷和 let-7a 验证了所提出的方法，检测限分别为 490 nM 和 26 pM，并显示出出色的特异性。我们相信这项工作开发了一种可行的方法，将 Cas12a 的抑制性适体用作生物传感目的的调节元件，丰富了基于 CRISPR/Cas12a 的分子诊断方法库，并促进了 CRISPR/Cas 适体的进一步开发和应用系统。

更新日期：2022-11-03

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