CRISPR/Cas12a\nCorona Nanomachine for Detecting Circulating\nTumor Nucleic Acids in Serum

Circulating tumor nucleic acids (CTNAs),\nwhich consist of cell-free\nDNA or RNA released from tumor cells, are utilized as potential biomarkers\nfor diagnosing and managing tumor prognosis. There is a significant\ndemand for developing a highly sensitive and reliable assay for CTNAs\ndetection. In this study, we engineered a CRISPR/Cas12a corona nanomachine\ncapable of detecting circulating tumor DNA and RNA in serum. This\nnanomachine consists of a protein shell incorporating Cas12a/crRNA\nribonucleoprotein complexes and a scaffold AuNP core decorated with\nsubstrate ssDNA strands. The protective CRISPR corona shields the\nnucleic acid core from degradation by nuclease DNase/RNase, thereby\nenhancing the stability of the CRISPR/Cas12a corona nanomachine in\nbiological fluids, even tolerating up to undiluted human serum and\nFBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated\nthrough the sequence-specific hybridization between crRNA and CTNAs.\nSubsequently, the activated CRISPR/Cas12a autonomously cleaves the\ncollateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled\nfragment and generating an increasing fluorescent signal. The CRISPR\ncorona nanomachine was successfully employed to detect various CTNAs,\nincluding circulating tumor (ct)DNA/RNA (<i>EGFR</i> L858R)\nand microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA\nand 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous\ndetection of both DNA and RNA in complex biological samples, offering\na promising tool for early diagnosis.