A Two-Step Strategy Based on Fpg Recognition and RCA-CRISPR/Cas12a for Ultrasensitive Detection of Gene Mutation

Abstract Accurate detection of gene mutations is crucial for acute myeloid leukaemia (AML) diagnosis and treatment. Although CRISPR/Cas systems enable sensitive mutation detection, their adaptability is constrained by dependence on restriction endonucleases. Herein, we propose a two-step strategy, termed Fpg-RCA-CRISPR/Cas12a for ultrasensitive detection of the p53 R175H, which integrates formamidopyrimidine DNA glycosylase (Fpg)-mediated recognition with rolling circle amplification (RCA)-coupled CRISPR/Cas12a technology. Validation confirmed that Fpg exhibits minimal activity when adenine (A) is opposite the damaged site. Based on this finding, we designed a padlock probe with a site-specific 8-oxoguanine (8-oxoG) modification. This modification enables selective recognition of G > A mutations. For the target TP53 c.524G > A (p53 R175H), the G > A substitution opposite the 8-oxoG site blocks Fpg cleavage, facilitating probe circularization and subsequent RCA. The resulting RCA products then activate the trans-cleavage activity of Cas12a, enabling cascade signal amplification and generating a strong fluorescence output. For wild-type p53, Fpg-mediated cleavage prevents circularization and amplification, yielding weak fluorescence. Taking advantage of the high specificity of the Fpg enzyme and the cascade amplification effect of the RCA−CRISPR/Cas12a system, this method achieves a limit of detection (LOD) of 0.29 fM and covers a linear range from 0.8 fM to 800 pM, along with high reproducibility, excellent selectivity, and negligible background interference. Notably, the assay can detect mutation frequencies as low as 0.1% in excess wild-type p53. Crucially, its clinical feasibility was confirmed by analyzing human serum samples, yielding results consistent with standard assays and thus demonstrating its practical reliability and translational potential.