Sensitive and visual detection of SMA using RPA-Cas12a one-step assay with ssDNA-modified crRNA

The irreversibility and lethality of Spinal Muscular Atrophy (SMA) underscore the urgency of newborn screening, as diagnostic delay in neonates causes irreversible motor neuron degeneration and poor outcomes. Current SMA detection methods are hindered by high costs, dependence on specialized equipment, and technical complexity, restricting their implementation in primary care setting. Here, we proposed a fast and sensitive SMA-(Recombinase Polymerase Amplification) RPA-Cas12a detection assay based on suboptimal protospacer adjacent motif (sPAM) and 3'-end ssDNA-modified crRNA, named SPSMC. The crRNA is designed based on the sPAM to enhance the specificity of SMN1 gene detection. The competition between RPA and Cas12a digestion for target DNA was resolved by using 3'-end ssDNA-modified crRNA. With ALB as a reference gene, this method can detect DNA at concentrations as low as 1.8 pM within 1 h. The sensitivity and specificity of the proposed method in differentiating SMA patients from non-SMA individuals were both 100 %. This strategy has been used for the detection of the SMN1 gene, which saves time, reduces contamination risks, and offers new possibilities for future point-of-care screening of SMA. In addition, the SPSMC system was successfully adapted to SMA lateral flow assay format and validated using 66 clinical samples, demonstrating 100 % sensitivity and specificity. The method is straightforward to perform, requires no bulky equipment, maintains full portability, and is more suitable for large-scale neonatal screening scenarios compared with traditional methods.