Regulating the trans -Cleavage Activity of CRISPR/Cas12a by Using an Elongation-Caged Single-Stranded DNA Activator and the Biosensing Applications

作者：Xinrui Fei, Chao Lei, Wei Ren, Xiaoling Liu, Chenghui Liu · 发表于：Analytical Chemistry · 年份：2023 · DOI：10.1021/acs.analchem.3c02471 · 被引用次数：37 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、RNA Interference and Gene Delivery

The CRISPR/Cas12a system exhibits extraordinary capability in the field of biosensing and molecular diagnosis due to its trans -cleavage ability. However, it is still desirable for precise control and programmable regulation of Cas12a trans -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 trans -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 trans -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 t...