Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care.

Rapid and reliable molecular diagnostics employing target nucleic acids and small biomarkers are crucial strategies required for the precise detection of numerous diseases. Although diagnoses based on nucleic acid recognition are some of the most efficient and precise procedures, these tests often require expensive equipment and skilled professionals. Recent advancements in diagnostic innovations, particularly those based on clustered regularly interspaced short palindromic repeats (CRISPR), aim to provide thorough screening at homes, in clinics, and in the field. In comparison to traditional molecular techniques like PCR, CRISPR/Cas-based detection, using the single-stranded nucleic acid trans-cleavage abilities of Cas12 or Cas13, shows significant potential as a molecular diagnostic tool. It offers benefits such as attomolar-level sensitivity, single-base precision, and rapid turnover rates. Both Cas enzymes demonstrate exceptional specificity and sensitivity, holding substantial promise in disease diagnostics and beyond. Consequently, various amplification-free CRISPR/Cas-based detection methods have emerged, aiming to maintain sensitivity despite the absence of pre-amplification. This allows for the detection of non-nucleic acid targets and facilitates integration into point-of-care settings. This Review highlights current advances in amplification-free CRISPR/Cas detection systems in disease diagnostics and investigates their utility in point-of-care settings. Furthermore, the mechanisms of alternative CRISPR-based amplification-free detection of other small molecules, aside from nucleic acids, for disease diagnosis will also be briefly discussed.