Zeng Muchu, Ke Yuqing, Zhuang Zhiyi, Qin Chao, Li Lai Yan, Sheng Gaoyuan, Li Zhuoru, Meng He, Ding Xianting
Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China.
Zhejiang University─University of Edinburgh Institute, Zhejiang University, Haining, Zhejiang 324400, PR China.
Anal Chem. 2022 Aug 2;94(30):10805-10812. doi: 10.1021/acs.analchem.2c01588. Epub 2022 Jul 20.
CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.
CRISPR相关（Cas）蛋白系统已越来越多地应用于核酸诊断。CRISPR/Cas12a在crRNA引导下靶向目标双链DNA（dsDNA）后，能够切割单链DNA（ssDNA），使其成为检测dsDNA的特异性工具。在核酸预扩增的辅助下，CRISPR/Cas12a能够实现阿托摩尔水平的dsDNA检测。然而，CRISPR/Cas12a中的这种强制预扩增还伴随着将预扩增产物转移到CRISPR/Cas12a系统中的额外步骤，这不仅繁琐、耗时，还会引发交叉污染风险。在此，我们展示了一种无需任何预扩增即可提高CRISPR/Cas12a系统灵敏度的多重crRNA策略。这种多重crRNA策略利用同一CRISPR/Cas12a系统中靶向同一dsDNA底物不同区域的多条crRNA序列。因此，检测信号无需扩增即可积累，从而提高了传统检测限。为了进行应用演示，以双链DNA病毒非洲猪瘟病毒（ASFV）的B646L基因为例。多重crRNA系统的检测限在无扩增的情况下可提高到约1皮摩尔（pM），比传统的单crRNA系统强约64倍。本研究中提出的多重crRNA系统稍作修改后，可推广到其他不易进行预扩增的生物传感场景。
