Development of Gap-Recombinase Polymerase Amplification Combined with CRISPR-Cas12a (Gap-RPA/CRISPR-Cas12a) for Rapid Screening of α0-Thalassemia Southeast Asian (--SEA) Deletion and Thai (--THAI) Deletion.

The α0-thalassemia Southeast Asian (--SEA) and Thai (--THAI) deletions comprise a significant global health burden, with high prevalence in Southeast Asia. Conventional methods like polymerase chain reaction (PCR) are limited in resource-poor settings due to complexity, time, and cost. This assay was developed as a rapid and simple option for point-of-care (POC) applications. Three assays specific for wild-type DNA, --SEA and --THAI deletions utilized recombinase polymerase amplification (RPA) for rapid DNA amplification at a constant low temperature of 37 °C, with a run time of 10 min, eliminating the need for a thermal cycler. The amplified products were detected using the highly specific CRISPR-Cas12a system and read visually via fluorescence or combined with a lateral flow assay (LFA) in 30 min. Successful engineering of a synthetic PAM site enabled Cas12a recognition of the --SEA target despite the absence of a suitable natural PAM. The platform demonstrated high accuracy in 74 blinded clinical samples, achieving a diagnostic accuracy of 98.20%, a sensitivity of 94.59%, and a specificity of 100% across 222 per-target evaluations. Detection from non-invasive samples achieved 80% to 100% accuracy, depending on the extraction method and readout format used. Our platform showed potential to meet several ASSURED criteria for diagnostic tests in resource-limited settings, demonstrating high potential for widespread use in genetic screening for α0-thalassemia (--SEA) and (--THAI).