Construction of luminescent bacteriophage using CRISPR technology and its application in Escherichia coli indentification
10.3760/cma.j.cn114452-20230908-00128
- VernacularTitle:基于CRISPR技术构建发光噬菌体及其在大肠埃希菌鉴定中的应用
- Author:
Minwei LI
1
;
Jing YAN
;
Hangyi LI
;
Zhiyun HAO
;
Zhong NI
;
Zhaoyang HU
;
Xiaorong WANG
;
Menghan XU
;
Chi WANG
;
Ruibing LI
;
Chengbin WANG
Author Information
1. 温州医科大学检验医学院生命科学学院临床检验诊断学2021级，温州 325035
- Keywords:
Bacteriophages;
Gene editing;
Escherichia coli;
Luciferases;
Homologous recombination
- From:
Chinese Journal of Laboratory Medicine
2024;47(4):437-443
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.
