An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification

• Published in: Biosensors & bioelectronics 2023-06, Vol.230, p.115248-115248, Article 115248
• Main Authors: Zhang, Yibin, Chen, Yong, Zhang, Qianling, Liu, Yizhen, Zhang, Xueji
• Format: Article
• Language: English
• Subjects: aM-level sensitive; Amplification-free; Biosensing Techniques; Cascade; COVID-19 - diagnosis; CRISPR-Cas Systems - genetics; CRISPR/Cas; Humans; Nucleic Acid Amplification Techniques; Nucleic Acids; RNA; SARS-CoV-2; SARS-CoV-2 - genetics
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2023.115248

The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus. •A novel cascade CRISPR-Dx system achieves aM-level sensitivity and detects SARS-CoV-2 standard within 20 min.•The cascade probe superimposes the Cas12a and Cas13a reactions, enabling detection without nucleic acid amplification.•This system reduces specialized equipment requirements and is suitable for developing countries.•The ASCas system is suitable for detecting other RNA targets and has potential applications in cancer diagnosis.