Advances in cascaded CRISPR for preamplification-free nucleic acid assays.
TL;DR
The design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades are introduced, including integration with digital detection platforms and AI-assistant algorithms are discussed.
Abstract
The development of rapid, sensitive, and specific nucleic acid assays is pivotal for advancing molecular detection in clinical diagnosis, food safety, and environmental monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems, renowned for their programmable and signal amplification capacity, have emerged as efficient tools for meeting these challenges. However, the intrinsic sensitivity of CRISPR/Cas assays relying on a single Cas effector is typically confined to the picomolar level, often necessitating complex nucleic acid preamplification. Cascaded CRISPR systems, which integrate sequential enzymatic reactions or multiple CRISPR effectors, can address this limitation by achieving nucleic acid preamplification-free signal enhancement. This review starts with the introduction of the design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades. Then, we highlight the deployment of these techniques across diverse bio-sensing scenarios, ranging from disease diagnosis to food and environmental surveillance. Finally, critical challenges and emerging frontiers are discussed, including integration with digital detection platforms and AI-assistant algorithms.