Cooperative effect of complementary antisense oligonucleotides and CRISPR effectors for universal DNA-based pathogen assay using nano-enabled colorimetry

Infectious diseases continue to pose significant challenges to global health, necessitating the development of diagnostic methodologies that are not only rapid and precise but also universally accessible. Herein, we developed a colorimetric CRISPR-Cas12a assay that utilizes CRISPR gene-editing technology, gold nanoparticles (AuNPs), and DNA hybridization principles to target a range of antibiotic-resistant and emerging pathogens, including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Candida auris (CA), and Human Papillomavirus (HPV). The assay utilizes specially engineered CRISPR-RNA (crRNA) and single-stranded oligonucleotides (ssDNAs) that correspond to a complementary single-stranded DNA sequence, termed the ’linker probe,’ which targets conserved genetic regions to enhance specificity and sensitivity. This design facilitates a simple visual readout through significant changes in the optical properties of AuNPs when the Cas12a enzyme, activated by target DNA, cleaves the linker probe. The assay, which does not require DNA amplification, demonstrated the ability to detect these pathogens with a detection limit of 7–8 copies/µL and achieved a clinical sensitivity of 100 % in detecting Chlamydia trachomatis and Neisseria gonorrhoeae from 60 de-identified clinical samples. Additionally, it directly detects DNA from these pathogens without the need for DNA extraction, employing a multiplexed lateral flow-based platform suitable for rapid clinical monitoring. This novel diagnostic approach offers significant improvements in the detection and management of infectious diseases, particularly in resource-limited settings, due to its simplicity, cost-effectiveness, and high specificity and sensitivity.