Zhao Shuang, Zhang Qiuting, Sun Jiudi, Li Shenghui, Wang Sheng, Zhou Dianming, Gong Xiaoqun
School of Life Sciences, Faculty of Medicine, Tianjin University and Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology (Tianjin), Tianjin 300072, China.
Key Laboratory of Post-Neuroinjury Neurorepair and Regeneration in Central Nervous System Ministry of Education in China and Tianjin, Tianjin Neurological Institute, Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052, China.
Nano Lett. 2025 May 21;25(20):8431-8441. doi: 10.1021/acs.nanolett.5c01939. Epub 2025 May 12.
CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for -cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.
CRISPR/Cas12a系统已成为分子诊断的多功能工具，但直接检测RNA和识别特定DNA点突变仍然具有挑战性。在此，我们报告了一种在间隔序列中有一个分裂位点的简单工程方法，可激活CRISPR/Cas12a（LbCas12a）进行切割，效率与野生型crRNA相似。工程化的crRNA通过用RNA片段替换3'端促进了RNA靶标的识别，增强了对单链DNA靶标的点突变特异性。基于此，我们实现了对微小RNA和DNA点突变的无扩增高灵敏度和特异性检测。为了进行临床样本验证，我们构建了反向荧光增强侧流试纸条（rLFTS），实现了飞摩尔水平的检测。此外，基于工程化crRNA的CRISPR/Cas12a系统还通过miRNA-21的细胞内和体内成像有效地识别肿瘤细胞。总之，这个工程化的crRNA平台增强了基于CRISPR/Cas12a的核酸检测，促进了其在分子诊断和生物成像中的广泛应用。
