RESUMO
CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in low-resource settings. Here, we utilized the chemically modified crRNA for the CRISPR-Cas-based assay (CM-CRISPR). We found that the extension and chemical modification to crRNA significantly enhanced the trans-cleavage activity of LbCas12a. The chemically modified crRNA was resistant to degradation, and CM-CRISPR showed superior detection capability in complex environments. CM-CRISPR could be combined with recombinase polymerase amplification (RPA) and applied in a droplet digital platform, enabling attomolar-level sensitivity. We also developed a portable and automated device for a digital CRISPR assay, which is amenable to point-of-care testing (POCT). The extraction-free procedure was integrated with this assay to streamline the workflow, and clinical samples were successfully detected. This work finds a simple and efficient way to improve the performance of CRISPR-Cas and develops a portable platform for POCT, representing a significant advance toward practical applications of CRISPR-based diagnostics.
RESUMO
Circular RNA (circRNA) and microRNA (miRNA) are both non-coding RNAs (ncRNAs) that serve as biomarkers for cancer diagnosis and prognosis. Quantitative detection of these ncRNAs is of particular importance to elucidate the functional mechanisms and evaluate their potential as biomarkers. However, the inherent structures of circRNA and miRNA are different from the mRNA, conventional qRT-PCR is unsuitable for the detection of these ncRNAs. Here, we propose a sensitive method for quantitative detection of circRNA and miRNA using polydisperse droplet digital CRISPR/Cas13a (PddCas13a). It can achieve limits of detection (LOD) as low as â¼10 aM without polymerase-based amplification. To efficiently detect the circRNA and miRNA in real samples, we use a chemically modified crRNA to enhance the stability of crRNA and improve the performance of Cas13a in complex environments containing contaminants. By integrating an extraction-free procedure with PddCas13a, we experimentally demonstrate the applicability of PddCas13a by testing clinical samples. Furthermore, we develop an automated and portable instrument for PddCas13a and verify its applicability for the detection of circRNA and miRNA from exosomes in point-of-care (POC) setting. This is the first report to detect the circRNA and miRNA simultaneously in POC setting. We envision this platform could promote the research of ncRNAs.
RESUMO
Rapid and precise nucleic acid testing at the point-of-care (POC) is essential for effective screening and management of infectious diseases. Current polymerase-based molecular diagnostics often suffer from potential cross-contamination issues, particularly in POC settings. Here, we introduce DECODE, a contamination-free nucleic acid detection platform integrating digital microfluidics (DMF) for nucleic acid extraction and a digital CRISPR amplification-free assay for pathogen detection. The digital CRISPR assay demonstrates sensitivity, detecting target DNA and RNA in the reaction mixture at concentrations of 10 and 5 copies/µL, respectively. Leveraging DMF-extracted samples enhances the performance of the digital CRISPR amplification-free assay. DECODE offers a sample-to-result workflow of 75 min using compact devices. Validation studies using clinical samples confirm DECODE's robust performance, achieving 100% sensitivity and specificity in detecting HPV18 from cervical epithelial cells and influenza A from nasal swabs. DECODE represents a versatile, contamination-free detection platform poised to enhance integrated public health surveillance efforts.