Cas9
清脆的
反式激活crRNA
计算生物学
DNA
基因组编辑
化学
核酸内切酶
聚合酶
分子生物学
生物
生物化学
基因
作者
Yuanzhao Hu,Yuefeng Qiao,Xiuqing Li,Zhenbo Xiang,Yi Wan,Peng Wang,Zhiqing Yang
出处
期刊:Talanta
[Elsevier]
日期:2023-12-01
卷期号:265: 124931-124931
标识
DOI:10.1016/j.talanta.2023.124931
摘要
Rapid, efficient, specific and sensitive diagnostic techniques are critical for selecting appropriate treatments for drug-resistant bacterial infections. To address this challenge, we have developed a novel diagnostic method, called the Dual-Cas Tandem Diagnostic Platform (DTDP), which combines the use of Cas9 nickase (Cas9n) and Cas12a. DTDP works by utilizing the Cas9n-sgRNA complex to create a nick in the target strand's double-stranded DNA (dsDNA). This prompts DNA polymerase to displace the single-stranded DNA (ssDNA) and leads to cycles of DNA replication through nicking, displacement, and extension. The ssDNA is then detected by the Cas12a-crRNA complex (which is PAM-free), activating trans-cleavage and generating a fluorescent signal from the fluorescent reporter. DTDP exhibits a high sensitivity (1 CFU/mL or 100 ag/μL), high specificity (specifically to MRSA in nine pathogenic species), and excellent accuracy (100%). The dual RNA recognition process in our method improves diagnostic specificity by decreasing the limitations of Cas12a in detecting dsDNA protospacer adjacent motifs (PAMs) and leverages multiple advantages of multi-Cas enzymes in diagnostics. This novel approach to pathogenic microorganism detection has also great potential for clinical diagnosis.
科研通智能强力驱动
Strongly Powered by AbleSci AI