Recent Advances in CRISPR/Cas Systems for Respiratory Pathogen Diagnostics

Early, rapid, and accurate detection is essential for clinical management and epidemiological control of acute respiratory infections caused by pathogens. Traditional testing methods such as microbial culture, serological testing, and PCR are restrictive in terms of operation and logistics and are therefore not easily used in point-of-care settings. The CRISPR/Cas system is an adaptive prokaryotic immune system composed of clustered regularly interspaced short palindromic repeats and their associated proteins, which has been used as a nucleic acid diagnostic platform with programmable sequence-specific target recognition and signal-amplifying collateral cleavage activity. Existing reviews have mostly focused on the classification of Cas enzymes or amplification strategies; in this review, a pathogen-centric approach was taken, covering viral pathogens (SARS-CoV-2, influenza virus, RSV, HAdV and VZV), bacterial pathogens (Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus) and fungal pathogens (Aspergillus fumigatus and Pneumocystis jirovecii). Key technological advances, such as isothermal amplification coupling, single-vessel integrated reaction designs, amplification-free digital detection, and electrochemical biosensor integration, are evaluated for Cas9-, Cas12-, and Cas13-based systems, with their mechanistic bases outlined. The current challenges that hinder clinical translation, such as sample matrix interference, multiple signal cross-talk, crRNA off-target effects, and the lack of large-scale validation studies, are critically assessed. At the same time, future pathways for portable, integrated, and inexpensive diagnostic platforms are suggested.