Knockout of human anxa6 gene in Caco-2 cells by CRISPR/Cas9 system
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    Abstract:

    [Objective] To study the interaction between Escherichia coli O157:H7 effector protein EspF and host ANXA6 protein and its pathogenic mechanism, we constructed a stable Caco-2 cell line with the knockout of anxa6 using CRISPR/Cas9 system.[Methods] We designed and synthesized three small guide RNA (sgRNA) which can specifically recognize anxa6 gene. We constructed Lenticrisprv2-sgRNA recombinant plasmid and transfected it into 293T cells to prepare sgRNA-Cas9 lentivirus. Then we infected Caco-2 cells with lentivirus, and applied puromycin to screen the positive cells. We isolated the monoclonal cells by limiting dilution and sequenced the cells to evaluate the knock-out of gene anxa6 and the off-target effect. Western blotting was employed to detect the expression level of ANXA6, cell counting kit 8 (CCK8) assay to determine cell proliferation, and immunofluorescence to detect the distribution of tight junction protein ZO-1. [Results] The anxa6 gene in Caco-2 cell line was knocked out, and no off-target effect in the 10 predicted sites was found. The knockout of anxa6 had no significant effect on cell proliferation. ZO-1 of Caco-2 and Caco-2anxa6‒/‒ cells displayed continuous distribution along the cell membrane, with complete structure. After transfection with EspF plasmids, the distribution of tight junction was incomplete with clear gaps and crack-like appearance. [Conclusion] We successfully constructed the Caco-2 cell line with the knock-out of anxa6. The cell line was used to preliminarily explore the role of ANXA6 protein in distribution of tight junction protein. This study provides an effective tool for exploiting the molecular mechanism of O157:H7 in mediating intestinal barrier injury through EspF-ANXA6 interaction.

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    [1] HOQUE M, RENTERO C, CAIRNS R, TEBAR F, ENRICH C, GREWAL T. Annexins-scaffolds modulating PKC localization and signaling[J]. Cellular Signalling, 2014, 26(6):1213-1225.
    [2] GREWAL T, ENRICH C. Annexins-modulators of EGF receptor signalling and trafficking[J]. Cellular Signalling, 2009, 21(6):847-858.
    [3] GERKE V, CREUTZ CE, MOSS SE. Annexins:linking Ca2+ signalling to membrane dynamics[J]. Nature Reviews Molecular Cell Biology, 2005, 6(6):449-461.
    [4] GERKE V, MOSS SE. Annexins:from structure to function[J]. Physiological Reviews, 2002, 82(2):331-371.
    [5] MOSS SE, MORGAN RO. The annexins[J]. Genome Biology, 2004, 5(4):219.
    [6] HUBER R, RÖMISCH J, PAQUES EP. The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes[J]. The EMBO Journal, 1990, 9(12):3867-3874.
    [7] SWAIRJO MA, ROBERTS MF, CAMPOS MB, DEDMAN JR, SEATON BA. Annexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties:31P-and 1H-NMR studies[J]. Biochemistry, 1994, 33(36):10944-10950.
    [8] GREWAL T, HOQUE M, CONWAY JRW, REVERTER M, WAHBA M, BEEVI SS, TIMPSON P, ENRICH C, RENTERO C. Annexin A6-A multifunctional scaffold in cell motility[J]. Cell Adhesion & Migration, 2017, 11(3):288-304.
    [9] GREWAL T, KOESE M, RENTERO C, ENRICH C. Annexin A6-regulator of the EGFR/Ras signalling pathway and cholesterol homeostasis[J]. The International Journal of Biochemistry & Cell Biology, 2010, 42(5):580-584.
    [10] ENRICH C, RENTERO C, de MUGA SV, REVERTER M, MULAY V, WOOD P, KOESE M, GREWAL T. Annexin A6-linking Ca2+ signaling with cholesterol transport[J]. Biochimica et Biophysica Acta:BBA-Molecular Cell Research, 2011, 1813(5):935-947.
    [11] TAKEDA M, LESER GP, RUSSELL CJ, LAMB RA. Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(25):14610-14617.
    [12] SWAGGART KA, DEMONBREUN AR, Vo AH, SWANSON KE, KIM EY, FAHRENBACH JP, HOLLEY-CUTHRELL J, ESKIN A, CHEN ZG, SQUIRE K, HEYDEMANN A, PALMER AA, NELSON SF, MCNALLY EM. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(16):6004-6009.
    [13] DEMONBREUN AR, QUATTROCELLI M, BAREFIELD DY, ALLEN MV, SWANSON KE, MCNALLY EM. An actin-dependent annexin complex mediates plasma membrane repair in muscle[J]. The Journal of Cell Biology, 2016, 213(6):705-718.
    [14] BABIYCHUK EB, MONASTYRSKAYA K, POTEZ S, DRAEGER A. Intracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells[J]. Cell Death & Differentiation, 2009, 16(8):1126-1134.
    [15] BRUYAND M, MARIANI-KURKDJIAN P, GOUALI M, de VALK H, KING LA, HELLO SL, BONACORSI S, LOIRAT C. Hemolytic uremic syndrome due to Shiga toxin-producing Escherichia coli infection[J]. Médecine et Maladies Infectieuses, 2018, 48(3):167-174.
    [16] EPPINGER M, CEBULA TA. Future perspectives, applications and challenges of genomic epidemiology studies for food-borne pathogens:a case study of enterohemorrhagic Escherichia coli (EHEC) of the O157:H7 serotype[J]. Gut Microbes, 2015, 6(3):194-201.
    [17] JERSE AE, YU J, TALL BD, KAPER JB. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87(20):7839-7843.
    [18] DEAN P, SCOTT JA, KNOX AA, QUITARD S, WATKINS NJ, KENNY B. The enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction[J]. PLoS Pathogens, 2010, 6(6):e1000961.
    [19] HUA Y, JU JW, WANG XY, ZHANG B, ZHAO W, ZHANG QW, FENG YZ, MA WB, WAN CS. Screening for host proteins interacting with Escherichia coli O157:H7 EspF using bimolecular fluorescence complementation[J]. Future Microbiology, 2018, 13:37-58.
    [20] HUA Y, WU JL, FU MQ, LIU JY, LI XX, ZHANG B, ZHAO W, WAN CS. Enterohemorrhagic Escherichia coli effector protein EspF interacts with host protein ANXA6 and triggers myosin light chain kinase (MLCK)-dependent tight junction dysregulation[J]. Frontiers in Cell and Developmental Biology, 2020, 8:613061.
    [21] ZHANG F. CRISPR-cas9:prospects and challenges[J]. Human Gene Therapy, 2015, 26(7):409-410.
    [22] PLIATSIKA V, RIGOUTSOS I. "Off-Spotter":very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs[J]. Biology Direct, 2015, 10(1):4.
    [23] GAYTÁN MO, MARTÍNEZ-SANTOS VI, SOTO E, GONZÁLEZ-PEDRAJO B. Type three secretion system in attaching and effacing pathogens[J]. Frontiers in Cellular and Infection Microbiology, 2016, 6:129.
    [24] DEY P. Targeting gut barrier dysfunction with phytotherapies:effective strategy against chronic diseases[J]. Pharmacological Research, 2020, 161:105135.
    [25] ZHANG F, WEN Y, GUO X. CRISPR/Cas9 for genome editing:progress, implications and challenges[J]. Human Molecular Genetics, 2014, 23(R1):R40-R46.
    [26] TREVINO AE, ZHANG F. Genome editing using Cas9 nickases[J]. Methods in Enzymology, 2014, 546:161-174.
    [27] 苏丛, 徐方明, 伍婷, 张鹏飞, 陈昊然, 刘艳艳, 兰燕虎, 李家斌, 律娜. 基于CRISPR-Cas9技术构建gpr41基因敲除的RAW264.7细胞系[J]. 安徽医科大学学报, 2020, 55(5):800-803. SU C, XU FM, WU T, ZHANG PF, CHEN HR, LIU YY, LAN YH, LI JB, LV N. Construction of RAW264.7 cell line with gpr41 gene knockout based on CRISPR-Cas9 technology[J]. Acta Universitatis Medicinalis Anhui, 2020, 55(5):800-803(in Chinese).
    [28] 姚叶豹, 王广菲, 董钦才, 曹诚, 刘萱. 利用CRISPR/Cas9系统构建HeLa细胞Cdc25C基因敲除稳定细胞株[J]. 军事医学, 2017, 41(5):359-362. YAO YB, WANG GF, DONG QC, CAO C, LIU X. Construction of stable Cdc25C knockout HeLa cell strains using CRISPR/Cas9 geneediting system[J]. Military Medical Sciences, 2017, 41(5):359-362(in Chinese).
    [29] NAEEM M, MAJEED S, HOQUE MZ, AHMAD I. Latest developed strategies to minimize the off-target effects in CRISPR-cas-mediated genome editing[J]. Cells, 2020, 9(7):1608.
    [30] 王远微, 刘雄. 基于CRISPR/Cas9技术构建TRPV1基因敲除的CACO-2稳定细胞系[J]. 西南民族大学学报(自然科学版), 2020, 46(3):241-249. WANG YW, LIU X. Construction of TRPV1 gene knockout Caco-2 stable cell line based on CRISPR/Cas9 technology[J]. Journal of Southwest Minzu University (Natural Science Edition), 2020, 46(3):241-249(in Chinese).
    [31] van BREEMEN RB, LI YM. Caco-2 cell permeability assays to measure drug absorption[J]. Expert Opinion on Drug Metabolism & Toxicology, 2005, 1(2):175-185.
    [32] KOMOR MA, BOSCH LJ, COUPÉ VM, RAUSCH C, PHAM TV, PIERSMA SR, MONGERA S, MULDER CJ, DEKKER E, KUIPERS EJ, van de WIEL MA, CARVALHO B, FIJNEMAN RJ, JIMENEZ CR, MEIJER GA, de WIT M. Proteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression[J]. The Journal of Pathology, 2020, 250(3):288-298.
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CHEN Hanzong, LIANG Song, LI Xinyue, FANG Yuting, PENG Yuejing, ZHANG Bao, ZHAO Wei, HUA Ying, WAN Chengsong. Knockout of human anxa6 gene in Caco-2 cells by CRISPR/Cas9 system. [J]. Acta Microbiologica Sinica, 2023, 63(3): 1217-1229

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History
  • Received:July 22,2022
  • Adopted:September 05,2022
  • Online: March 08,2023
  • Published: March 04,2023
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