Advances in Amplification-Free Nucleic Acid Detection via CRISPR-Cas System: From Cascade Circuits to Advanced Readouts

Abstract

While enzymatic amplification remains essential for achieving ultrasensitive detection in molecular diagnostics,amplification-free detection methods offer distinct advantages in simplicity, speed, and cost-effectiveness, particularly for point-ofcareapplications. Their value is particularly evident in scenarios where enzymatic amplification is not a viable option. This includesresource-limited settings that lack sophisticated equipment, as well as applications such as live-cell imaging, where amplification isinherently unfeasible. However, the widespread adoption of these techniques has been hindered by limited sensitivity. Recently,clustered regularly interspaced short palindromic repeats (CRISPR)-Cas have been adapted into a powerful platform for moleculardiagnostics, providing a promising solution to this sensitivity bottleneck. By leveraging the efficient signal amplification generatedby the trans-cleavage activity of Cas12 or Cas13, these systems achieve remarkable sensitivity within a simple, isothermal format. This review summarizes recent advances in amplification-free nucleic acid detection using the CRISPR-Cas system, beginning witha fundamental overview of key Cas proteins and then systematically describing how Cas12 and Cas13 contribute differently todetection, reflecting their unique characteristics. By summarizing these advances, this review aims to provide valuable insights forthe future development and application of CRISPR-based amplification-free biosensors.