Xinping Wang

The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system’s specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings. CRISPR-Cas12a nucleic acid detection largely involves pre-amplification and is limited in its ability to directly detect RNA. Here, authors present a split Cas12a system that enables direct, highly sensitive, and cost-effective detection of both DNA and RNA without amplification.

The CRISPR/Cas12a system has revolutionized molecular diagnostics, yet the direct detection of RNA, particularly those with complex structures, remains a significant challenge. Here, we present an updated SCas12a system, termed SCas12aV2, which enables precise, amplification-free detection of highly structured RNA molecules. By optimizing the length of scaffold RNA, targeting asymmetric structures, and utilizing dsDNA-ssDNA hybrid activators, we have significantly reduced steric hindrance in the detection system, thereby markedly enhancing both sensitivity and kinetics compared to traditional DNA activators. The SCas12aV2 assay achieves a detection limit of 246 aM for pooled activators and 10 pM for single-site targeting, demonstrating high specificity for single nucleotide polymorphisms (SNPs). It successfully identifies viable bacteria and SARS-CoV-2 infections in clinical samples. The assay is versatile and can be applied to various Cas12a orthologs, including thermostable CtCas12a. This work advances molecular diagnostics by improving the accuracy and efficiency of RNA detection.

A novel sodium alginate/gellan gum-based composite film with mulberry anthocyanins (MAE) as an indicator was developed for real-time pork freshness monitoring. Research showed that the incorporation of MAE significantly improved the physical properties of the composite films, increasing the tensile strength (25.2 %), elongation at break (24.6 %), and thickness (30.4 %). FT-IR, X-RD, and SEM analyses indicate that the compatibility and molecular interactions between MAE and polymer matrix have been enhanced. The composite films exhibited pH-responsive behavior, demonstrating distinct color changes in buffer solutions across a pH range of 2–12. When used to evaluate the freshness of pork, the composite film will show a significant color change, which is consistent with the texture of the pork. The color difference of the films showed a strong linear relationship with the storage time of the pork, total volatile basic nitrogen (TVB-N), and pH. Therefore, composite films showed great potential for application as smart packaging in monitoring pork freshness. • Mulberry anthocyanins (MAE) significantly improve the mechanical properties of sodium alginate/gellan gum (SA/GG) films. • A noticeable colour change (ΔE >12) can be observed with the naked eye, transitioning from red (pH 2) to yellow (pH 12). • The strong correlation (R 2 > 0.92) between film color and spoilage indicators of pork enables effective spoilage detection.

Abstract The CRISPR/Cas12a system has revolutionized molecular diagnostics; however, its application in directly detecting complex structured RNA remains challenging. Recently, we have developed a RNA detection method called SCas12a, which exhibits high sensitivity and efficiency in detecting RNA molecules devoid of intricate secondary structures. Here, we present an enhanced SCas12a assay (SCas12aV2) that facilitates precise amplification-free detection of highly structured RNA molecules. Our approach reengineers the split Cas12a system by optimizing the scaffold RNA length and targeting asymmetric RNA structures, thereby minimizing steric hindrance. We observe that utilization of a dsDNA-ssDNA hybrid DNA activator significantly enhances both the sensitivity and kinetics compared to those achieved using traditional ssDNA or dsDNA activators. The SCas12aV2 assay demonstrates exceptional sensitivity, with a limit of detection reaching 246 aM for pooled activators and 10 pM for single-site targeting. It also exhibits high specificity for single nucleotide polymorphisms (SNPs) and successfully identifies viable bacterial populations and SARS-CoV-2 infections from clinical samples. The assay’s versatility is further highlighted by its applicability to various Cas12a orthologs, including the thermostable CtCas12a. This work offers a significant advance in molecular diagnostics, enhancing the potential for accurate and efficient RNA detection.