Thermal-enhanced atypical suboptimal PAM-dependent CRISPR/Cas12a amplification for detecting BCR1-Type PML::RARα fusion and L217F mutation.

Current clinical methods for detecting low-abundance PML::RARα fusion and drug-resistant single-base mutations face significant challenges, highlighting the urgent need for a rapid, sensitive, cost-effective genetic detection method for diagnosing acute promyelocytic leukemia. The CRISPR/Cas12a system has emerged as a revolutionary tool in nucleic acid detection. However, its applications are frequently constrained by the requirement for protospacer adjacent motifs (PAMs). In this study, we demonstrated that a moderate temperature increase enhanced CRISPR/Cas12a activity while allowing the use of atypical suboptimal PAMs. Utilizing a PCR-driven, suboptimal PAM-dependent CRISPR/Cas12a cascade, we sensitively detected the BCR1-type PML::RARα fusion with a detection limit of 0.1 aM. This method effectively identified low-abundance mutant cells in the presence of wild-type cells and demonstrated its efficacy in detecting minimal residual disease samples from patients with relapsed acute promyelocytic leukemia. Additionally, we developed a sensitive approach for detecting L217F single-base mutations using a cascade strategy of polymerase chain reaction, ligase chain reaction and CRISPR/Cas12a, achieving a detection limit of 10 aM in various wild-type DNA samples. Consequently, these methods hold considerable promise for the rapid diagnosis of acute promyelocytic leukemia, the sensitive detection of and the low-abundance drug-resistance nucleic acid mutations.