Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing

作者：Qingnan Li, Dongxia Wang, Gui‐Mei Han, Bo Liu, An‐Na Tang, De‐Ming Kong · 发表于：Analytical Chemistry · 年份：2023 · DOI：10.1021/acs.analchem.3c03131 · 被引用次数：48 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、Advanced Memory and Neural Computing

The trans -cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in “on–off–on” mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the “RESET” effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer–substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 μM, and 8.3 × 10 –5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. More...