sb9b00209_si_001.pdf (1.35 MB)
HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation
journal contribution
posted on 2019-09-30, 15:03 authored by Linxian Li, Shiyuan Li, Na Wu, Jiacheng Wu, Gang Wang, Guoping Zhao, Jin WangThe
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.