Fei Xinrui, Lei Chao, Ren Wei, Liu Xiaoling, Liu Chenghui
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, P. R. China.
College of Chemistry and Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China.
Anal Chem. 2023 Aug 15;95(32):12169-12176. doi: 10.1021/acs.analchem.3c02471. Epub 2023 Aug 2.
The CRISPR/Cas12a system exhibits extraordinary capability in the field of biosensing and molecular diagnosis due to its -cleavage ability. However, it is still desirable for precise control and programmable regulation of Cas12a -cleavage activity to promote the in-depth studies and application expansion of Cas12a-based sensing platforms. In this work, we have developed a new and robust CRISPR/Cas12a regulation mechanism by endowing the activator with the function of caging crRNA ingeniously. Specifically, we constructed an integrated elongation-caged activator (EL-activator) by extending the ssDNA activator on the 3'-end. We found that appending only about 8 nt that is complementary to the crRNA repeat region is enough to cage the crRNA spacer/repeat region, thus effectively inhibiting Cas12a -cleavage activity. The inner inhibition mechanism was further uncovered after a thorough investigation, demonstrating that the EL-activator works by impeding the conformation of crRNA required for Cas12a recognition and destroying its affinity with Cas12a. By further switching on the elongated moiety on the EL-activator using target biomarkers, the blocked -cleavage activity of Cas12a can be rapidly recovered. Finally, a versatile sensing platform was established based on the EL-activator regulation mechanism, expanding the conventional Cas12a system that only directly recognizes DNA to the direct detection of enzymes and RNA biomarkers. This work has enriched the CRISPR/Cas12a regulation toolbox and expanded its sensing applications.
由于其切割能力，CRISPR/Cas12a系统在生物传感和分子诊断领域展现出非凡的能力。然而，为了促进基于Cas12a的传感平台的深入研究和应用扩展，对Cas12a切割活性进行精确控制和可编程调节仍然是很有必要的。在这项工作中，我们通过巧妙地赋予激活剂封闭crRNA的功能，开发了一种全新且强大的CRISPR/Cas12a调节机制。具体而言，我们通过在3'端延伸单链DNA激活剂构建了一种整合的延伸封闭激活剂（EL-激活剂）。我们发现，仅附加约8个与crRNA重复区域互补的核苷酸就足以封闭crRNA间隔区/重复区域，从而有效抑制Cas12a切割活性。经过深入研究，进一步揭示了其内在抑制机制，表明EL-激活剂通过阻碍Cas12a识别所需的crRNA构象并破坏其与Cas12a的亲和力来发挥作用。通过使用目标生物标志物进一步开启EL-激活剂上的延伸部分，Cas12a被阻断的切割活性可以迅速恢复。最后，基于EL-激活剂调节机制建立了一个通用的传感平台，将仅直接识别DNA的传统Cas12a系统扩展到对酶和RNA生物标志物的直接检测。这项工作丰富了CRISPR/Cas12a调节工具箱并扩展了其传感应用。
