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.

基于 CRISPR 的分子检测已显示出在护理点进行早期癌症诊断和传染病检测的巨大潜力。在这里，我们研究了延长 CRISPR RNA (crRNA) 的 5' 末端对 CRISPR 检测的影响，并开发了一个 5' eNd EXTension CRISPR（称为“NEXT CRISPR”）生物传感平台，用于快速、高灵敏度和特异性核酸检测。通过利用 5' 末端延伸的 crRNA 和优化的 CRISPR 反应系统，我们展示了增强的 NEXT CRISPR 检测。NEXT CRISPR 与重组酶聚合酶扩增 (RPA) 兼容，并且可以轻松适应荧光检测和横向流条读数。为了启用即时检测， 分钟。此外，我们通过检测临床拭子样本对我们的 NEXT CRISPR 生物传感平台进行了临床验证，实现了与传统 PCR 方法相当的性能。这种快速、灵敏且经济实惠的 NEXT CRISPR 生物传感平台为癌症和传染病的即时诊断提供了一种很有前景的工具。