Li Linxian, Li Shiyuan, Wu Na, Wu Jiacheng, Wang Gang, Zhao Guoping, Wang Jin
University of Chinese Academy of Sciences , Beijing , 100049 , China.
Key Laboratory of Synthetic Biology , Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , 200032 , China.
ACS Synth Biol. 2019 Oct 18;8(10):2228-2237. doi: 10.1021/acssynbio.9b00209. Epub 2019 Sep 30.
The next-generation CRISPR-based molecular diagnostics has the merits of rapidness, accuracy, and portability. We discovered the Cas12a -cleavage activity against collateral single-stranded DNA (ssDNA) and employed the activity to develop a rapid nucleic acid detection system, namely HOLMES (one-hour low-cost multipurpose highly efficient system). Here, with the employment of thermophilic CRISPR-Cas12b, we create HOLMESv2 for four different applications: (1) specifically discriminating single nucleotide polymorphism (SNP); (2) simply detecting virus RNA, human cell mRNA and circular RNA; (3) conveniently quantitating target nucleic acids with a one-step system combined with LAMP amplification in a constant temperature, thus avoiding cross-contamination; (4) accurately quantitating target DNA methylation degree with the combination of Cas12b detection and bisulfite treatment. These results highlight the potential of HOLMESv2 as a promising platform for both molecular diagnostics and epigenetics applications.
基于CRISPR的新一代分子诊断技术具有快速、准确和便携的优点。我们发现了Cas12a对单链DNA（ssDNA）的切割活性，并利用该活性开发了一种快速核酸检测系统，即HOLMES（一小时低成本多功能高效系统）。在此，通过使用嗜热CRISPR-Cas12b，我们创建了适用于四种不同应用的HOLMESv2：（1）特异性鉴别单核苷酸多态性（SNP）；（2）简单检测病毒RNA、人类细胞mRNA和环状RNA；（3）通过结合恒温LAMP扩增的一步系统方便地定量目标核酸，从而避免交叉污染；（4）通过结合Cas12b检测和亚硫酸氢盐处理准确地定量目标DNA甲基化程度。这些结果突出了HOLMESv2作为分子诊断和表观遗传学应用的一个有前景平台的潜力。
