Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay

N6-Methyladenosine (m 6 A) 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 CRISPRm 6 A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m 6 A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m 6 A RNA with single-base resolution. Compared to the CRISPRm 6 A assay using Cas13a alone, the tandem CRISPRm 6 A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m 6 A. Particularly, the proposed assay enabled quantification of m 6 A 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 CRISPRm 6 A assay supports two key applications in biological samples: (1) precise determination of m 6 A sites and (2) quantitative measurement of m 6 A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.