Western blotting及序列测序表明anxa6基因敲除单克隆细胞构建成功;脱靶效应评估结果显示预测的10个脱靶位点均无脱靶现象;基因敲除对细胞增殖能力无明显影响;免疫荧光结果显示,Caco-2及Caco-2anxa6‒/‒细胞紧密连接蛋白ZO-1均沿细胞膜连续分布,结构完整,转染EspF质粒后出现分布不完整、缺口等现象。
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.
CRISPR/Cas9系统anxa6EspF蛋白Caco-2细胞紧密连接CRISPR/Cas9 systemanxa6EspF proteinsCaco-2 cellstight junction广东省自然科学基金2018B030311063广东省基础与应用基础研究基金2021A1515011240国家自然科学基金32100143广东省自然科学基金(2018B030311063);广东省基础与应用基础研究基金(2021A1515011240);国家自然科学基金(32100143)the Natural Science Foundations of Guangdong Province2018B030311063the Guangdong Basic and Applied Basic Research Foundation2021A1515011240the National Natural Science Foundation of China32100143This work was supported by the Natural Science Foundations of Guangdong Province (2018B030311063), the Guangdong Basic and Applied Basic Research Foundation (2021A1515011240), and the National Natural Science Foundation of China (32100143)
LentiCRISPRv2-sgRNA vector validation results. A: PCR agarose gel validation of Stbl3 bacterial broth containing recombinant plasmids. Marker: DL2000 DNA molecular weight standard; R1-1‒R3-2 indicate different colony clones corresponding to the three recombinant LentiCRISPRv2-sgRNA plasmids were transformed into each of them. B−D: The sequence results of Homo-ANXA6-sg1, Homo-ANXA6-sg2 and Homo-ANXA6-sg3.
Western blotting to verify the expression of ANXA6 protein. Lane 1−4: sgRNA1-Cas9 lentivirus-infected Caco-2 monoclonal cells; WT: Normal Caco-2 cells.
DNA sequencing results. A: Sequencing results of normal Caco-2 cells. B: Sequencing results of ANXA6-sgRNA1-1. C: Deletion mutation of monoclonal cellular anxa6 gene in Caco-2 cells. Red markers are the initially designed sgRNA sequences; "–" represents the bases missing after mutation.
Distribution of EspF protein disrupting tight junction protein ZO-1 (60×oil microscope). Green is the fluorescence expressed by plasmid, red is ZO-1 protein, blue is DAPI-stained nucleus. A: ZO-1 distribution after transfection of Caco-2 cells with pEGFP (E) and pEGFP-EspF (EE) plasmids. B: ZO-1 distribution of Caco-2 cells stably knocked down with pEGFP (E) and pEGFP-EspF (EE) plasmids after transfection with pEGFP (E) and pEGFP-EspF (EE) plasmids. C: Fluorescence intensity analysis of ZO-1 in Caco-2 cells. D: Fluorescence intensity analysis of ZO-1 in Caco-2 cells stably knocked down with anxa6 gene. Error bars represent means±SD from three independent experiments; Asterisks indicate P values (***: P < 0.001).
GERKE V, MOSS SEAnnexins: from structure to function200282233137110.1152/physrev.00030.2001
GERKE V, MOSS SE. Annexins: from structure to function[J]. Physiological Reviews, 2002, 82(2): 331-371.
MOSS SE, MORGAN ROThe annexins20045421910.1186/gb-2004-5-4-219
MOSS SE, MORGAN RO. The annexins[J]. Genome Biology, 2004, 5(4): 219.
HUBER R, RÖMISCH J, PAQUES EPThe crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes19909123867387410.1002/j.1460-2075.1990.tb07605.x
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.
SWAIRJO MA, ROBERTS MF, CAMPOS MB, DEDMAN JR, SEATON BAAnnexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties: 31P- and 1H-NMR studies19943336109441095010.1021/bi00202a013
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.
GREWAL T, HOQUE M, CONWAY JRW, REVERTER M, WAHBA M, BEEVI SS, TIMPSON P, ENRICH C, RENTERO CAnnexin A6-A multifunctional scaffold in cell motility2017113288304
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.
GREWAL T, KOESE M, RENTERO C, ENRICH CAnnexin A6-regulator of the EGFR/Ras signalling pathway and cholesterol homeostasis2010425580584
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.
ENRICH C, RENTERO C, de MUGA SV, REVERTER M, MULAY V, WOOD P, KOESE M, GREWAL TAnnexin A6—linking Ca2+ signaling with cholesterol transport20111813593594710.1016/j.bbamcr.2010.09.015
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.
TAKEDA M, LESER GP, RUSSELL CJ, LAMB RAInfluenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion200310025146101461710.1073/pnas.2235620100
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.
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 EMAnnexin A6 modifies muscular dystrophy by mediating sarcolemmal repair2014111166004600910.1073/pnas.1324242111
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.
DEMONBREUN AR, QUATTROCELLI M, BAREFIELD DY, ALLEN MV, SWANSON KE, MCNALLY EMAn actin-dependent annexin complex mediates plasma membrane repair in muscle2016213670571810.1083/jcb.201512022
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.
BABIYCHUK EB, MONASTYRSKAYA K, POTEZ S, DRAEGER AIntracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells200916811261134
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.
BRUYAND M, MARIANI-KURKDJIAN P, GOUALI M, de VALK H, KING LA, HELLO SL, BONACORSI S, LOIRAT CHemolytic uremic syndrome due to Shiga toxin-producing Escherichia coli infection201848316717410.1016/j.medmal.2017.09.012
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.
EPPINGER M, CEBULA TAFuture perspectives, applications and challenges of genomic epidemiology studies for food-borne pathogens: a case study of enterohemorrhagic Escherichia coli (EHEC) of the O157: H7 serotype20156319420110.4161/19490976.2014.969979
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.
JERSE AE, YU J, TALL BD, KAPER JBA genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells199087207839784310.1073/pnas.87.20.7839
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.
DEAN P, SCOTT JA, KNOX AA, QUITARD S, WATKINS NJ, KENNY BThe enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction201066e100096110.1371/journal.ppat.1000961
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.
HUA Y, JU JW, WANG XY, ZHANG B, ZHAO W, ZHANG QW, FENG YZ, MA WB, WAN CSScreening for host proteins interacting with Escherichia coli O157: H7 EspF using bimolecular fluorescence complementation201813375810.2217/fmb-2017-0087
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.
HUA Y, WU JL, FU MQ, LIU JY, LI XX, ZHANG B, ZHAO W, WAN CSEnterohemorrhagic Escherichia coli effector protein EspF interacts with host protein ANXA6 and triggers myosin light chain kinase (MLCK)-dependent tight junction dysregulation2020861306110.3389/fcell.2020.613061
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.
ZHANG FCRISPR-cas9: prospects and challenges201526740941010.1089/hum.2015.29002.fzh
ZHANG F. CRISPR-cas9: prospects and challenges[J]. Human Gene Therapy, 2015, 26(7): 409-410.
PLIATSIKA V, RIGOUTSOS I"Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs2015101410.1186/s13062-015-0035-z
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.
GAYTÁN MO, MARTÍNEZ-SANTOS VI, SOTO E, GONZÁLEZ-PEDRAJO BType three secretion system in attaching and effacing pathogens20166129
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.
DEY PTargeting gut barrier dysfunction with phytotherapies: effective strategy against chronic diseases202016110513510.1016/j.phrs.2020.105135
DEY P. Targeting gut barrier dysfunction with phytotherapies: effective strategy against chronic diseases[J]. Pharmacological Research, 2020, 161: 105135.
ZHANG F, WEN Y, GUO XCRISPR/Cas9 for genome editing: progress, implications and challenges201423R1R40R4610.1093/hmg/ddu125
ZHANG F, WEN Y, GUO X. CRISPR/Cas9 for genome editing: progress, implications and challenges[J]. Human Molecular Genetics, 2014, 23(R1): R40-R46.
TREVINO AE, ZHANG FGenome editing using Cas9 nickases2014546161174
TREVINO AE, ZHANG F. Genome editing using Cas9 nickases[J]. Methods in Enzymology, 2014, 546: 161-174.
SU C, XU FM, WU T, ZHANG PF, CHEN HR, LIU YY, LAN YH, LI JB, LV NConstruction of RAW264.7 cell line with gpr41 gene knockout based on CRISPR-Cas9 technology2020555800803
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).
YAO YB, WANG GF, DONG QC, CAO C, LIU XConstruction of stable Cdc25C knockout HeLa cell strains using CRISPR/Cas9 geneediting system2017415359362
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).
NAEEM M, MAJEED S, HOQUE MZ, AHMAD ILatest developed strategies to minimize the off-target effects in CRISPR-cas-mediated genome editing202097160810.3390/cells9071608
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.
WANG YW, LIU XConstruction of TRPV1 gene knockout Caco-2 stable cell line based on CRISPR/Cas9 technology2020463241249
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).
van BREEMEN RB, LI YMCaco-2 cell permeability assays to measure drug absorption200512175185
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.
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 MProteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression20202503288298
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.