MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, PR China.
Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, PR China.
Anal Chem. 2021 Feb 2;93(4):2038-2044. doi: 10.1021/acs.analchem.0c03708. Epub 2021 Jan 7.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the -cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the -cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.
成簇规律间隔短回文重复序列（CRISPR）和 CRISPR 相关（Cas）（CRISPR/Cas）系统由于其高保真度、可编程性和高效信号放大能力，创新了下一代生物传感器。开发基于 CRISPR/Cas 的可视化检测系统有助于即时生物标志物诊断。现有的基于 CRISPR/Cas9 的可视化检测方法受到 Cas9 固有特性的限制。在此，我们探索了 Cas13a 对带有核糖核苷酸的 DNA 寡核苷酸的 -切割能力，消除了后续聚合反应中 -切割底物的不可用性，并开发了一种均相基于 CRISPR/Cas13a 的可视化检测系统（称为 vCas），用于 miRNA 的特异性和灵敏检测。结果表明，vCas 可以提供 1 fM 的 miR-10b 检测限，具有单碱基特异性，并且可用于分析血清和细胞提取物中的 miRNA。总之，vCas 在临床分子诊断方面具有广阔的应用前景。
