机构：[1]State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, Department of Pulmonary and Critical Care Medicine, Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, West China Hospital, Sichuan University, Chengdu 610041, China.四川大学华西医院[2]College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, China.[3]West China-California Research Center for Predictive Intervention Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.四川大学华西医院[4]Frontiers Medical Center, Tianfu Jincheng Laboratory, Chengdu 610212, China.[5]School of Chemical Engineering, Sichuan University, Chengdu 610065, China.
N6-Methyladenosine (m6A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm6A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m6A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m6A RNA with single-base resolution. Compared to the CRISPRm6A assay using Cas13a alone, the tandem CRISPRm6A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m6A. Particularly, the proposed assay enabled quantification of m6A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm6A assay supports two key applications in biological samples: (1) precise determination of m6A sites and (2) quantitative measurement of m6A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.
基金：
National Key Research
and Development Program of China (2022YFF1103000),
National Natural Science Foundation of China (22074100),
Postdoctoral Fellowship Program of CPSF (GZC20231774),
Natural Science Foundation of Sichuan (2025ZNSFSC1041),
and Chengdu Science and Technology Support Program
(2024-YF05-00697-SN).
语种：
外文
PubmedID：
中科院(CAS)分区：
出版当年[2025]版：
大类|1 区化学
小类|1 区分析化学
最新[2025]版：
大类|1 区化学
小类|1 区分析化学
第一作者：
第一作者机构：[1]State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, Department of Pulmonary and Critical Care Medicine, Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, West China Hospital, Sichuan University, Chengdu 610041, China.
共同第一作者：
通讯作者：
通讯机构：[2]College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, China.[5]School of Chemical Engineering, Sichuan University, Chengdu 610065, China.
推荐引用方式(GB/T 7714)：
Shi Kunyu,Zhang Yong,Tao Yiran,et al.Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay[J].Analytical Chemistry.2025,doi:10.1021/acs.analchem.4c06782.
APA：
Shi Kunyu,Zhang Yong,Tao Yiran,Wang Yuxi,Yang Jinliang...&Yang Hao.(2025).Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.Analytical Chemistry,,
MLA：
Shi Kunyu,et al."Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay".Analytical Chemistry .(2025)
化学