期刊
ACS SYNTHETIC BIOLOGY
卷 8, 期 10, 页码 2228-2237出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00209
关键词
CRISPR; Cas12b; nucleic acid detection; DNA methylation quantitation; HOLMES
资金
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDB19040200]
- ToloBio Future Innovation and Development Program [2018-02]
- National Natural Science Foundation of China [31922046]
The next-generation CRISPR-based molecular diagnostics has the merits of rapidness, accuracy, and portability. We discovered the Cas12a trans-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.
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