Framework‐Hotspot Enhanced Trans Cleavage of CRISPR‐Cas12a for Clinical Samples Detection

作者：Fengqin Li, Junru Li, Weiqiang Yang, Shuai Yang, Congshuo Chen, Ling Du, Junyang Mei, Qianyun Tang, Xiaojing Chen, Chi Yao, Dayong Yang, Xiaolei Zuo, Peifeng Liu · 发表于：Angewandte Chemie International Edition · 年份：2023 · DOI：10.1002/anie.202305536 · 被引用次数：63 · 研究领域：CRISPR and Genetic Engineering、Innovation and Socioeconomic Development、Advanced biosensing and bioanalysis techniques

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.