CRISPR/Cas12a coupled with MIRA: A specific and rapid assay for human DNA in challenging forensic matrices

Human DNA detection is crucial in forensic medicine, particularly for trace, degraded, or mixed samples, which demand high sensitivity, specificity, and rapid processing. Traditional methods, such as immunological assays and PCR-based techniques, often suffer from operational complexity, limited sensitivity, or high equipment dependency. To address these challenges, we developed a novel detection system combining multienzyme isothermal rapid amplification (MIRA) with CRISPR-Cas12a for the rapid, specific, and portable human DNA identification. By targeting the human mitochondrial cytochrome b (CYTB) gene and sex-determining Region Y(SRY) gene, we designed MIRA primers and CRISPR-Cas12a crRNA to enable dual recognition and signal amplification. The method involves isothermal amplification at 37°C followed by CRISPR-Cas12a-mediated cleavage, producing detectable fluorescence or lateral flow chromatographic signals. Our system achieves ultra-sensitive detection and high specificity, distinguishing human DNA from non-human sources (e.g., pig, chicken, mouse), and also enables accurate gender identification, further enhancing its utility in forensic and genetic studies. Compared to traditional qPCR, this approach demonstrates superior sensitivity, faster turnaround (≤ 45 min), and minimal equipment requirements, making it ideal for forensic applications. Moreover, the blood, mixed, and degraded samples were used to confirm its robustness, with results interpretable via blue-light fluorescence or colloidal gold test strips. In summary, the MIRA-CRISPR/Cas12a system overcomes the limitations of conventional techniques, offering a rapid, cost-effective, and reliable solution for forensic human DNA identification, with potential extensions to wildlife conservation and food safety testing.