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  

Construction of luminescent bacteriophage using CRISPR technology and its application in Escherichia coli indentification

作　　者：李民伟 颜菁 李航逸 郝治云 倪忠 胡朝阳[4] 王笑蓉 许梦寒 王驰[3] 李芮冰 王成彬[1] Li Minwei;Yan Jing;Li Hangyi;Hao Zhiyun;Ni Zhong;Hu Zhaoyang;Wang Xiaorong;Xu Menghan;Wang Chi;Li Ruibing;Wang Chengbin(Clinical Laboratory Diagnostics Class of 2021,Wenzhou Medical University,Wenzhou 325035,China;Department of Veterinary Medicine,University of Cambridge,Cambridge CB30ES,UK;Department of Clinical Laboratory,The First Medical Center,Chinese PLA General Hospital,Beijing 100853,China;School of Life Sciences,Biology Class of 2021,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]温州医科大学检验医学院生命科学学院,温州325035 [2]剑桥大学兽医系,英国剑桥CB30ES [3]解放军总医院第一医学中心检验科,北京100853 [4]江苏大学生命科学学院,镇江212013

出　　处：《中华检验医学杂志》2024年第4期437-443,共7页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(8217080305)。

摘　　要：目的构建针对大肠埃希菌的重组发光噬菌体(HT7),并评估其对大肠埃希菌的鉴定能力。方法首先通过基因工程构建小向导RNA表达质粒pCRISPR-sg(1~10)和PFN-1000同源重组质粒菌株,并用双层平板筛选切割效率最高的小向导RNA(sgRNA);采用同源重组与规律成簇的间隔短回文重复(CRISPR)系统联合介导的基因编辑技术,将Nanoluc萤光素酶基因整合入T7噬菌体10A基因下游的非编码区,并成功构建发光噬菌体HT7;通过噬菌斑实验和液体扩增实验评估HT7噬菌体与T7噬菌体生物特性差异;此外用2022年1月至2023年6月解放军总医院第一医学中心临床采集分离的51株大肠埃希菌、20株肺炎克雷伯菌、14株金黄色葡萄球菌、6株屎肠球菌、5株粪肠球菌、3株鲍曼不动杆菌和1株铜绿假单胞菌评估HT7噬菌体的检测专一性和检出限。结果构建的10个CRISPR靶向切割系统中,sgRNA8靶标的切割效率最高,成斑效率为0.18;噬菌体经pCas9/pCRISPR/PFN-10003质粒系统3轮重组筛选后,PCR验证均为2798 bp的重组噬菌体条带,说明成功构建了HT7噬菌体。重组噬菌体在裂解效率(P<0.001)、一步生长曲线(P=0.001)、感染复数(P=0.031)的生物特性分析中,均有显著差异,裂解暴发点和对数生长节点均延长了10 min,最佳感染复数为0.1;临床样本测试,可识别裂解6株大肠埃希菌,其余菌株均不裂解,4.5 h内可检出低于10 CFU/ml的病原菌。结论成功开发了一种高效的噬菌体基因编辑系统,并成功构建发光噬菌体HT7,该噬菌体在4.5 h内能特异性检测出低于10 CFU/ml的大肠埃希菌,且对其他菌株无裂解作用,显示出良好的检测专一性和低检出限。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 2798 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

关 键 词：细菌噬菌体 基因编辑 大肠杆菌 萤光素酶类 同源重组 

分 类 号：R446.5[医药卫生—诊断学]