STAR-CRISPR: a one-pot ultraspecific CRISPR strategy for rapid, visualized SNV detection and genotyping in point-of-care diagnostics.

Single nucleotide variation (SNV), as a key biomarker for disease diagnosis and personalized treatment, faces challenges in rapid and accurate detection. This study developed a single-tube accelerated recognition of SNVs strategy named STAR-CRISPR, which could accomplish SNV detection within only 20 min. This method integrated isothermal amplification and CRISPR/Cas12b cleavage system in one pot, and results could be directly identified by the naked eye. This method could accurately distinguish single-base differences, and could detect as low as 1% mutations against high background interference. We verified the proposed method by testing 70 clinical samples of idiopathic chronic pancreatitis, pancreatic cancer and acute myeloid leukemia. Results showed 100% consistency with next-generation sequencing results, demonstrating good accuracy and reliability of the proposed method. To further facilitate point-of-care diagnosis, we developed integrated miniature microfluidic chips, which greatly simplified sample identification and enabled logical interpretation of results. The combined STAR-CRISPR and microfluidic platform not only identifies SNVs but also supports simultaneous visual genotyping of wild-type, homozygous, and heterozygous mutations. Consequently, the proposed strategy is accurate, rapid, and versatile, holding significant potential for next-generation molecular diagnostics.