HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation
HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation
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DOI：
10.1021/acssynbio.9b00209
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发表时间：
2019-10-01
影响因子：
4.7
通讯作者：
Wang, Jin
中科院分区：
文献类型：
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作者：
Li, Linxian;Li, Shiyuan;Wang, Jin
下一代基于CRISPR的分子诊断具有快速、准确和便携的优点。我们发现了Cas 12 a对侧链单链DNA（ssDNA）的反式切割活性，并利用该活性开发了快速核酸检测系统，即HOLMES（一小时低成本多用途高效系统）。在此，我们利用嗜热CRISPR-Cas 12 b构建了HOLMESv 2，用于四种不同的应用：（1）特异性区分单核苷酸多态性（SNP）;（2）简单检测病毒RNA、人细胞mRNA和环状RNA;（3）在恒温条件下结合LAMP扩增的一步系统方便地定量靶核酸，从而避免交叉污染;（4）在特定的环境中检测靶核酸。（4）结合Cas 12 b检测和亚硫酸氢盐处理准确定量靶DNA甲基化程度。这些结果突出了HOLMESv 2作为分子诊断和表观遗传学应用的有前途的平台的潜力。
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.