Bai Xiaopeng, Gao Panqi, Qian Keli, Yang Jiandong, Deng Haijun, Fu Tiwei, Hu Yuan, Han Miaomiao, Zheng Huizhi, Cao Xiaoxia, Liu Yuliang, Lu Yaoqin, Huang Ailong, Long Quanxin
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China.
Department of Infection Control, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Front Microbiol. 2022 May 13;13:847373. doi: 10.3389/fmicb.2022.847373. eCollection 2022.
CRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.
A combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.
A set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated , the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.
These results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.
据报道，基于CRISPR-Cas13a系统的核酸检测方法具有快速且灵敏的DNA检测能力。然而，能够实现CRISPR-Cas13a对人类病原体进行单碱基分辨率DNA检测的crRNA筛选策略仍不明确。
提出了一种将合理设计与靶向突变锚定的CRISPR RNA（crRNA）筛选策略相结合的方法。
发现一组crRNA能够使CRISPR-Cas13系统显著区分临床分离菌株中氟喹诺酮耐药突变与高度同源的野生型，不同突变位点的信号比在8.29至38.22之间。对于使用临床分离株的基因组DNA评估临床性能，与基于培养的表型分析相比，特异性和灵敏度分别为100%和91.4%。
这些结果表明，CRISPR-Cas13a系统在调整crRNA后具有用于单核苷酸多态性（SNP）检测的潜力。我们相信这种crRNA筛选策略将被广泛用于早期耐药性监测和临床治疗指导。
