Fig. 3: Detection of multiple NPM1 mutations via an enAsU-R Cas12a-induced in vitro cleavage assay.

a PAM identification assay of enAsU-R Cas12a. Normalized cleavage rates of all possible 4-base PAMs are shown at different blue depths. b Cleavage efficiency comparison of enAs, enAsU, and enAsU-R Cas12a on PAM TTTV (n = 3 targets), NVCA (n = 12 targets), RVCC (n = 6 targets), and NNCG (n = 16 targets), respectively. P values are determined by two-tailed Student’s t-tests, ns., no significance. c Proportions of different NPM1 gene c.863_864 4-bp insertion types in 2348 NPM1-mutated AML patients. The most common 11 types are marked in different colours. d The genomic location of the c.863_864 4-bp insertion is shown above and marked by a red triangle. The blue blocks represent exons of the NPM1 gene. The mutant DNA sequence and six designed crRNAs are shown below, with the inserted bases marked in red and PAMs marked in blue. The PAM is not included in the synthesized crRNA. N = A/C/T/G. e Comparison of 36 Cas12a/crRNA pairs by detecting 2E10 copies of insTCTG and WTDNA substrates, reacting for 30 min at 37 °C. Values and error bars reflect the means and standard deviation (s.d.) of three biological replicates. The chessboard diagrams display the final relative fluorescence intensity, and the histogram shows the fluorescence intensity ratio (TCTG/WT). f Flowchart of the TA clone-based mismatch tolerance assay. g The six chessboard diagrams display the normalized fluorescence intensity of six Cas12a/crRNA1-induced in vitro detection of the PCR purified products of 32 random TA clones, WT, and NC. h The normalized fluorescence intensity of six Cas12a/crRNA1-induced in vitro detection of the top 11 NPM1 gene c.863_864 4-bp insertions using 5E10 copies of DNA as substrates, and reacting for 20 min at 37 °C. The fluorescence image shows the naked-eye result of enAsU-R under a blue light. Source data are provided as a Source Data file.