利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

doi:10.16431/j.cnki.1671-7236.2020.03.003

Abstract

Abstract: The aim of this study was to use the CRISPR/Cas9 and λ-Red cascade technology to perform a seamless knockout the heat-labile toxin (LT) gene of enterotoxigenic Escherichia coli (ETEC) K88 and obtain a K88 LT deficient strain.The homologous sequences of boundary sequences at both ends were obtained by sequence alignment,and a donor fragment was constructed containing the LT boundary sequences,the chloromycetin selection marker,sgRNA sequences and the LT homology arm.The donor fragment was transformed into ETEC K88,and LT gene was knocked out using λ-Red homologous recombination system and CRISPR/Cas9 gene editing system,respectively.The K88 LT-deficient strain was obtained by PCR,and the hemolysis ability and growth curve of the knockout strain were determined.The results showed that LT gene was successfully replaced by the corresponding donor fragment using λ-Red homologous recombination system,and the chloromycetin selection marker was efficiently deleted using CRISPR/Cas9 gene editing system.The gene editing system successfully performed a seamless knockout of LT gene of ETEC K88.In vitro experiment results showed that the K88 LT deficient strain had no capacity to hemolysis and grew slowly than the wild-type strain.It indicated that LT affected the pathogenicity and growth performance of the strain.Therefore,the results suggested that λ-Red and CRISPR/Cas9 cascade knockout methods could be used for knockout of LT toxin genes,and could also be used for other E.coli knockouts.The construction of K88 LT-strain laid the foundation for further study on the pathogenesis of LT toxin.

Key words: enterotoxigenic Escherichia coli(ETEC); CRISPR/Cas9; λ-Red recombination system; heat-labile enterotoxin

CLC Number:

S852.61⁺2

TAN Keqin, MA Xianyong, CUI Yiyan, TIAN Zhimei, DENG Dun. Construction of Enterotoxigenic E.coli LT Knockout Strain Using CRISPR/Cas9 and λ-Red Cascaded Technology[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 666-675.

References

[1] WANG H,ZHONG Z,LUO Y,et al.Heat-stable enterotoxins of enterotoxigenic Escherichia coli and their impact on host immunity[J].Toxins,2019,11(1):24-36.
[2] SIMON N C,KLAUS A,BARBIERI J T.Novel bacterial ADP-ribosylating toxins:Structure and function[J].Nature Reviews Microbiology,2014,12(9):599-611.
[3] MONTAGUE T,CRUZ J,GAGNON J,et al.CHOPCHOP:A CRISPR/Cas9 and TALEN web tool for genome editing[J].Nucleic Acids Research,2014,42(1):401-407.
[4] 杨佳儒,郭美华,柳爱华,等.CRISPR/Cas9的原理及其应用进展[J].昆明医科大学学报,2016,37(5):118-122. YANG J R,GUO M H,LIU A H,et al.Priciple and application progress of CRISPR-Cas9[J].Journal of Kuming Medical University,2016,37(5):118-122.(in Chinese)
[5] MA Y,ZHANG L,UANG X.Genome modification by CRISPR/Cas9[J].The FEBS Journal,2014,281(23):5186-5193.
[6] DOETSCHMAN T,GEORGIEVA T.Gene editing with CRISPR/Cas9 RNA-directed nuclease[J].Circulation Research,2017,120(5):876-894.
[7] LIU S C,LU H H,FAN H C,et al.The identification of the TRPM8 channel on primary culture of human nasal epithelial cells and its response to cooling[J].Medicine,2017,96(31):e7640.
[8] LEE Z H,YAMAGUCHI N,ITO T.Using CRISPR/Cas9 system to introduce targeted mutation in Arabidopsis,in plant transcription factors:Methods and Protocols[N].NewYork:Springer,2018.
[9] 余深翼,赵金荣,郑玲红,等.利用CRISPR/Cas9技术构建大肠杆菌aroA基因的敲除系统及其初步应用[J].畜牧兽医学报,2016,47(4):762-770. YU S Y,ZHAO J R,ZHENG L H,et al.The application of CRISPR/Cas9 technology for aroA gene knockout in Escherichia coli[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):762-770.(in Chinese)
[10] ZHANG W P,BERBEROV E M,JESSICA F,et al.Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies[J].Infection & Immunity,2006,74(6):3107-3114.
[11] YANG J J,SUN B B,HUANG H,et al.High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and Ⅰ-SceⅠ cleavage[J].Applied & Environmental Microbiology,2014,80(13):3826-3834.
[12] SHANKAR S,SREEKUMAR A,PRASAD D,et al.Genome editing of oncogenes with ZFNs and TALENs:Caveats in nuclease design[J].Cancer Cell International,2018,18(1):169-180.
[13] BEUMER K J,TRAUTMAN J K,CHRISTIAN M,et al.Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila[J].G3-Genes Genomes Genetics,2013,3(10):1717-1725.
[14] 郭梓茹,张立,马云龙,等.CRISPR-Cas9介导的蒙古牛MSTN基因敲除细胞系的建立[J].中国畜牧兽医,2018,45(4):841-849. GUO Z R,ZHANG L,MA Y L,et al.Establishment of MSTN gene knockdown in Mongolia bovine cell lines mediated by CRISPR-Cas9 system[J].China Animal Husbandry & Verterinary Medicine,2018,45(4):841-849.(in Chinese)
[15] DEMIRCI Y,ZHANG B,UNVER T.CRISPR/Cas9:An RNA-guided highly precise synthetic tool for plant genome editing[J].Journal of Cellular Physiology,2017,233(3):1844-1859.
[16] MIRHOSEINI A,AMANI J,NAZARIAN S.Review on pathogenicity mechanism of enterotoxigenic Escherichia coli and vaccines against it[J].Microbial Pathogenesis,2018,117:162-169.
[17] DUBREUIL J D,ISAACSON R E,SCHIFFERLI D M.Animal enterotoxigenic Escherichia coli[J].Ecosal Plus,2016,7(1):1-47.
[18] KAPER J B,NATARO J P,MOBLEY H L.Pathogenic Escherichia coli[J].Nature Reviews Microbiology,2004,295(6):355-356.
[19] WEIGLMEIER P R,RÖSCH P,BERKNER H.Cure and curse:E.coli heat-stable enterotoxin and its receptor guanylyl cyclase C[J].Toxins,2010,2(9):2213-2229.
[20] VON M A,TOBIAS J,WIKLUND G,et al.Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC)[J].Scientific Reports,2017,7(1):12514-12524.
[21] NAGY B,FEKETE P Z.Enterotoxigenic Escherichia coli in veterinary medicine[J].Journal of Medical Microbiology,2005,295(6):443-454.
[22] VERBRUGGHE E,VAN P A,LEYMAN B,et al.Heat-labile enterotoxin of Escherichia coli promotes intestinal colonization of Salmonella enterica[J].Comparative Immunology,Microbiology and Infectious Diseases,2015,43:1-7.
[23] 余树培.K88ac⁺大肠杆菌减毒株的构建及其在小鼠体内的初步应用[D].扬州:扬州大学,2016. YU S P.Construction of K88ac⁺ ETEC attenuated strain and its preliminary application in vivo of mouse[D].Yangzhou:Yangzhou University,2016.(in Chinese)
[24] 鲁曦,傅恩清,潘蕾,等.肠毒性大肠杆菌gspD基因敲除以及对LT毒素分泌的影响[J].基因组学与应用生物学,2015,34(12):2538-2543. LU X,FU N Q,PAN L,et al.Deletion of gspD gene in chromosome of enterotoxigenic Escherichia coli enteritis and the effect on LT toxin secretion[J].Genomics and Applied Biology,2015,34(12):2538-2543.(in Chinese)
[25] 付瑞,刘华,段勇,等.应用siRNA技术抑制产肠毒素大肠杆菌LT基因表达的研究[J].国际检验医学杂志,2015,36(8):1009-1011. FU R,LIU H,DUAN Y,et al.Inhibition of LT gene expression in enterotoxin-producing Escherichia coli by siRNA[J].International Journal of Laboratory Medicine,2015,36(8):1009-1011.(in Chinese)
[26] HAINES S,ARNAUD-BARBE N,PONCET D,et al.IscR regulates synthesis of colonization factor antigen Ⅰ fimbriae in response to iron starvation in enterotoxigenic Escherichia coli[J].Journal of Bacteriology,2015,197(18):2896-2907.
[27] 刘变芳,殷先华,吕欣,等.λ-Red重组系统敲除肠出血性大肠杆菌O157:H7细胞毒素stx2基因[J].中国兽医学报,2010,30(7):926-930. LIU B F,YIN X H,LYU X,et al.Knockout stx2 gene of enterohemorrhagic E.coli O157:H7 by λ-Red recombination system[J].Chinese Journal of Veterinary Science,2010,30(7):926-930.(in Chinese)
[28] 夏军,郑明刚,王玲,等.运用CRISPR/Cas系统敲除大肠杆菌磷酸烯醇式丙酮酸羧化酶基因及其对脂肪酸代谢的影响[J].微生物学通报,2016,43(8):1864-1871. XIA J,ZHENG M G,WANG L,et al.Knocking out phosphoenolpyruvate carboxylase gene by CRISPR/Cas and its influence on fatty acid metabolism in Escherichia coli[J].Microbiology China,2016,43(8):1864-1871.(in Chinese)
[29] ZHANG Y,GE X,YANG F,et al.Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells[J].Scientific Reports,2014,4:5405-5409.
[30] THOMASON L C,COSTANTINO N,COURT D L.Examining a DNA replication requirement for bacteriophage λ Red-and Rac prophage RecET-promoted recombination in Escherichia coli[J].MBio,2016,7(5):1-16.
[31] 杨正时,钟熙.平板免疫溶血法检测产毒性大肠杆菌不耐热肠毒素[J].医学研究杂志,1987,16(8):13-14. YANG Z S,ZHONG X.Detection of toxigenic Escherichia coli heat-labile enterotoxin by plate immunohemolytic method[J].Journal of Medical Research,1987,16(8):13-14.(in Chinese)
[32] CURTIS T,TAKEUCHI I,GRAM L,et al.The influence of the toxin/antitoxin mazEF on growth and survival of Listeria monocytogenes under stress[J].Toxins,2017,9(1):31-47.
[33] ZHAO J,WANG Q,LI M,et al.Escherichia coli toxin gene hipA affects biofilm formation and DNA release[J].Microbiology,2013,159(3):633-640.
[34] HWANG H H,YANG F J,CHENG T F,et al.The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens[J].Phytopathology,2013,103(9):888-899.

Construction of Enterotoxigenic E.coli LT Knockout Strain Using CRISPR/Cas9 and λ-Red Cascaded Technology

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