NEXT CRISPR: An enhanced CRISPR-based nucleic acid biosensing platform using extended crRNA

CRISPR-based molecular detection has shown great potential for early cancer diagnostics and infectious disease testing at the point of care. Here, we investigated the effect of extending the 5′ end of CRISPR RNA (crRNA) on CRISPR detection and developed a 5′ eNd EXTension CRISPR (termed “NEXT CRISPR”) biosensing platform for rapid, highly sensitive, and specific nucleic acid detection. By taking advantage of a 5′ end-extended crRNA and optimized CRISPR reaction system, we demonstrated an enhanced NEXT CRISPR detection. The NEXT CRISPR is compatible with recombinase polymerase amplification (RPA), and can be easily adapted for both fluorescence detection and lateral flow strip readout. To enable point-of-care testing, we coupled RPA/NEXT CRISPR with a lateral flow assay to develop a NEXT CRISPR biosensing platform for human papillomavirus (HPV) 16 DNA detection with attomolar sensitivity within 30 min. Further, we clinically validated our NEXT CRISPR biosensing platform by detecting clinical swab samples, achieving a comparable performance with the conventional PCR method. This rapid, sensitive, and affordable NEXT CRISPR biosensing platform provides a promising tool for point-of-care diagnostics of cancers and infectious diseases.