Citation: Hao Hu, Zhengxin Ye, Lei Zhang, Kejun Dong, Bei Yan, Longjie Li, Wei Zhang, Xianjin Xiao. A PAM-free and universal CRISPR-Cas12a activation model for ultra-sensitive DNA methylation detection[J]. Chinese Chemical Letters, ;2026, 37(1): 111184. doi: 10.1016/j.cclet.2025.111184 shu

A PAM-free and universal CRISPR-Cas12a activation model for ultra-sensitive DNA methylation detection

Hao Hu ^{a, 1} ,
Zhengxin Ye ^{a, 1} ,
Lei Zhang ^{a, 1} ,
Kejun Dong ^{a, c} ,
Bei Yan ^a ,
Longjie Li ^{d, *,} ,
Wei Zhang ^{b, c, *,} ,
Xianjin Xiao ^{a, e, *,}

a.
Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
b.
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
c.
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
d.
School of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, China
e.
Laboratory of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

lilongjie@whpu.edu.cn

weizhang_dr@sina.com

xiaoxianjin@hust.edu.cn

Received Date: 28 October 2024
Revised Date: 6 February 2025
Accepted Date: 7 April 2025
Available Online: 8 April 2025

Figures(5)

DNA methylation is an important promising biomarker for cancer diagnosis and monitoring. Therefore, the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer. However, it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample. In this work, we proposed a protospacer adjacent motif (PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method. Through recognizing the dsDNA with toehold region, CRISPR-Cas12a not only got rid of the limitation of PAM, but also improved its distinction ability for single CpG site methylation, nearly 5-fold that of conventional PAM-containing dsDNA. We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single CpG methylation site. Our results showed that the discrimination factor was > 200. Then, we constructed toe-dsDNA by using "heating and freezing", which made our method universally applicable and feasible. In addition, we greatly simplified the difficulty of primer design. Our method detected four highly methylated genes acyl carrier protein (ACP), CLV3/ESR-related (CLE), Disabled (DAB) and Homeobox (HOX) with a detection limit of 0.01% and excellent linearity in DNA methylation standards. Then, we verified the clinical utility of this method in 29 hepatocellular carcinomas, 11 ovarian cancers and 4 health people. In conclusion, we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method, which achieves high congruence in sensitivity, specificity and universality, fully demonstrating its significant clinical application value.
- CRISPR-Cas12a,
- DNA methylation,
- PAM-free,
- Ultra-sensitive,
- Toe-dsDNA
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沈阳化工大学材料科学与工程学院沈阳 110142