meHOLMES: A CRISPR-cas12a-based method for rapid detection of DNA methylation in a sequence-independent manner
Songkuan Zhuang,
Tianshuai Hu,
Xike Zhou,
Hongzhong Zhou,
Shiping He,
Jie Li,
Long Qiu,
Yuehui Zhang,
Yong Xu,
Hao Pei,
Dayong Gu,
Jin Wang
Affiliations
Songkuan Zhuang
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China; Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen 518060, China
Tianshuai Hu
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China
Xike Zhou
The Fifth People's Hospital of Wuxi, Affiliated to Jiangnan University, Wuxi, Jiangsu 214007, China
Hongzhong Zhou
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China
Shiping He
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China
Jie Li
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China
Long Qiu
Tolo Biotechnology Co., Ltd, Wuxi, Jiangsu 214174, China
Yuehui Zhang
Shenzhen Bao An Peoples Hospital, Shenzhen 518060, China
Yong Xu
Department of Clinical Laboratory, Shenzhen Third People's Hospital, The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, National Clinical Research Center for Infectious Disease, Shenzhen 518112, China
Hao Pei
The Fifth People's Hospital of Wuxi, Affiliated to Jiangnan University, Wuxi, Jiangsu 214007, China; Corresponding author.
Dayong Gu
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China; Corresponding author.
Jin Wang
Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China; Tolo Biotechnology Co., Ltd, Wuxi, Jiangsu 214174, China; Corresponding author. Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China.
Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner.
