CRISPR/Cas9技术敲除斑马鱼lkb1基因及表型分析
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摘要
目的利用CRISPR/Cas9技术敲除斑马鱼lkb1基因,观察和验证纯合子表型,初步探讨Lkb1对肾小管发育的影响。方法针对斑马鱼lkb1基因设计并体外合成gRNA,通过显微注射技术将gRNA和Cas9 mRNA注入野生型胚胎一细胞期卵黄中,利用T7E1酶切和测序筛选出高效gRNA,通过测序筛选出F0代突变体及可遗传突变体,野生鱼和可遗传突变体鱼杂交产生F1代杂合子突变体,测序鉴定F1代杂合子突变类型,通过F1代杂合子自交产生F2代纯合子幼鱼,通过观察鉴定出纯合子表型;通过RT-PCR和免疫印迹验证纯合子表型。结果制备了斑马鱼lkb1 gRNA和Cas9 mRNA,筛选出3种产生移码突变的杂合子,发现斑马鱼lkb1基因敲除纯合子幼鱼受精后第5天游动时头尾剧烈摆动,异常兴奋,鱼鳔缺失,体轴弯曲,卵黄消耗过快,肝脏暗淡,肠道水肿;第9天大多数死亡;纯合子g6pase、pdk2b、pcpck1 mRNA显著升高,Lkb1完全不表达;肾小管标志蛋白mRNA显著升高。结论利用CRISPR/Cas9技术敲除斑马鱼lkb1基因产生了多种表现,包括肝脏衰竭、肠道水肿、体轴弯曲、鱼鳔缺失、早期致死的表型,同时表明Lkb1对斑马鱼肾小管发育有影响。
Objective To knock out lkb1 gene in zebrafish by CRISPR/Cas9 technique, observe and verify homozygote phenotype, and preliminarily explore the effect of lkb1 on the development of renal tubule. Methods The gRNA targeting lkb1 gene was designed and synthesized. The gRNA and Cas9 mRNA were injected into the wild-type cell-phase embryo yolk by microinjection technique. The high-efficiency gRNA was screened by T7 E1 digestion and sequencing. The F0 mutants and heritable mutants were screened by sequencing. Wild-type fish and heritable mutant fish were used to hybridize to produce F1 hybrid heterozygous mutants, and F1 hybrid heterozygous mutants were identified by sequencing. Then F2 generation homozygous juveniles were produced by self-crossing of F1 heterozygotes. Homozygous phenotypes were identified, and homozygous phenotypes were verified by qPCR and Western blot analysis. Results The zebrafish lkb1 gRNA and Cas9 mRNA were prepared, and 3 heterozygotes with frameshift mutations were obtained. It was found that the zebrafish Lkb1 knockout homozygous juveniles violently swayed at the head and tail on the 5 th day after fertilization, and with missed swim bladder, bended body axis, fast yolk consumption, dim liver, and edematous intestinal tract, and most of them died on the 9 th day. The mRNA levels of homozygous g6 pase, pdk2 b and pcpck1 were significantly increased, while lkb1 was not expressed at all. The mRNA levels of homozygous renal markers, trmp7, slc12 a1 and slc12 a3 were also obviously elevated. Conclusion Knockout of zebrafish lkb1 gene by CRISPR/Cas9 technology results in liver failure, edematous intestinal tract, bended body axis, missed swim bladder, and early lethal phenotype. The knockout also shows effect on the development of renal tubule.
Objective To knock out lkb1 gene in zebrafish by CRISPR/Cas9 technique, observe and verify homozygote phenotype, and preliminarily explore the effect of lkb1 on the development of renal tubule. Methods The gRNA targeting lkb1 gene was designed and synthesized. The gRNA and Cas9 mRNA were injected into the wild-type cell-phase embryo yolk by microinjection technique. The high-efficiency gRNA was screened by T7 E1 digestion and sequencing. The F0 mutants and heritable mutants were screened by sequencing. Wild-type fish and heritable mutant fish were used to hybridize to produce F1 hybrid heterozygous mutants, and F1 hybrid heterozygous mutants were identified by sequencing. Then F2 generation homozygous juveniles were produced by self-crossing of F1 heterozygotes. Homozygous phenotypes were identified, and homozygous phenotypes were verified by qPCR and Western blot analysis. Results The zebrafish lkb1 gRNA and Cas9 mRNA were prepared, and 3 heterozygotes with frameshift mutations were obtained. It was found that the zebrafish Lkb1 knockout homozygous juveniles violently swayed at the head and tail on the 5 th day after fertilization, and with missed swim bladder, bended body axis, fast yolk consumption, dim liver, and edematous intestinal tract, and most of them died on the 9 th day. The mRNA levels of homozygous g6 pase, pdk2 b and pcpck1 were significantly increased, while lkb1 was not expressed at all. The mRNA levels of homozygous renal markers, trmp7, slc12 a1 and slc12 a3 were also obviously elevated. Conclusion Knockout of zebrafish lkb1 gene by CRISPR/Cas9 technology results in liver failure, edematous intestinal tract, bended body axis, missed swim bladder, and early lethal phenotype. The knockout also shows effect on the development of renal tubule.
引文
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[13] DING F,HU Q,WANG M,et al.Knockout of SlSBPASE suppresses carbon assimilation and alters nitrogen metabolism in tomato plants[J].Int J Mol Sci,2018,19(12):E4046.DOI:10.3390/ijms19124046.
[14] RAMANAGOUDR-BHOJAPPA R,CARRINGTON B,RAMASWAMI M,et al.Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth,sexual development and fertility[J].PLoS Genet,2018,14(12):e1007821.DOI:10.1371/journal.pgen.1007821.
[15] 刘菁,梁森,袁志.CRISPR/Cas9技术敲除faf1基因对斑马鱼软骨及肌节发育的影响[J].第三军医大学学报,2017,39(17):1709-1714.DOI:10.16.16/j.10000-5404.201612182.LIU J,LIANG S,YUAN Z.Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development[J].J Third Mil Med Univ,2017,39(17):1709-1714.DOI:10.16.16/j.10000-5404.201612182.
[16] KUANG X,LIU C,FANG J,et al.The tumor suppressor gene lkb1 is essential for glucose homeostasis during zebrafish early development[J].FEBS Lett,2016,590(14):2076-2085.DOI:10.1002/1873-3468.12237.
[17] ZHANG F,WEN Y,GUO X.CRISPR/Cas9 for genome editing:progress,implications and challenges[J].Hum Mol Genet,2014,23(R1):R40-R46.DOI:10.1093/hmg/ddu125.
[18] NGUYEN H B,BABCOCK J T,WELLS C D,et al.LKB1 tumor suppressor regulates AMP kinase/mTOR-independent cell growth and proliferation via the phosphorylation of Yap[J].Oncogene,2013,32(35):4100-4109.DOI:10.1038/onc.2012.431.
[19] LI J,LIU J,LI P P,et al.Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion[J].J Exp Clin Cancer Res,2014,33:70.DOI:10.1186/s13046-014-0070-0.
[20] SUN A,LI C,CHEN R,et al.GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells[J].Prostate,2016,76(2):172-183.DOI:10.1002/pros.23106.
[21] MARTIN S G,ST JOHNSTON D.A role for drosophila LKB1 in anterior-posterior axis formation and epithelial polarity[J].Nature,2003,421(6921):379-384.DOI:10.1038/nature01296.
[22] SHELLY M,POO M M.Role of LKB1-SAD/MARK pathway in neuronal polarization[J].Dev Neurobiol,2011,71(6):508-527.DOI:10.1002/dneu.20884.
[23] KWON S K,SANDO R,LEWIS T L,et al.LKB1 regulates mitochondria-dependent presynaptic calcium clearance and neurotransmitter release properties at excitatory synapses along cortical axons[J].PLoS Biol,2016,14(7):e1002516.DOI:10.1371/journal.pbio.1002516.
[2] YADAV H,DEVALARAJA S,CHUNG S T,et al.TGF-β1/smad3 pathway targets PP2A-AMPK-FoxO1 signaling to regulate hepatic gluconeogenesis[J].J Biol Chem,2017,292(8):3420-3432.DOI:10.1074/jbc.M116.764910.
[3] QIU B J,WEI W,ZHU J W,et al.EMT induced by loss of LKB1 promotes migration and invasion of liver cancer cells through ZEB1-induced YAP signaling[J].Oncol Lett,2018,16(5):6465-6471.DOI:10.3892/ol.2018.9445.
[4] YAO Y H,CUI Y,QIU X N,et al.Attenuated LKB1-SIK1 signaling promotes epithelial-mesenchymal transition and radioresistance of non-small cell lung cancer cells[J].Chin J Cancer,2016,35:50.DOI:10.1186/s40880-016-0113-3.
[5] HONG B,ZHANG J,YANG W,et al.Activation of the LKB1-SIK1 signaling pathway inhibits the TGF-β-mediated epithelial-mesenchymal transition and apoptosis resistance of ovarian carcinoma cells[J].Mol Med Rep,2018,17(2):2837-2844.DOI:10.3892/mmr.2017.8229.
[6] HU M,ZHAO T K,LIU J,et al.Decreased expression of LKB1 is associated with epithelial-mesenchymal transition and led to an unfavorable prognosis in gastric cancer[J].Hum Pathol,2019,83:133-139.DOI:10.1016/j.humpath.2018.08.017.
[7] LIN H,LI N,HE H,et al.AMPK inhibits the stimulatory effects of TGF-β on Smad2/3 activity,cell migration,and epithelial-to-mesenchymal transition[J].Mol Pharmacol,2015,88(6):1062-1071.DOI:10.1124/mol.115.099549.
[8] SHORNING B Y,JARDé T,MCCARTHY A,et al.Intestinal renin-angiotensin system is stimulated after deletion of Lkb1[J].Gut,2012,61(2):202-213.DOI:10.1136/gutjnl-2011-300046.
[9] MCGHEE A,SIVARAJAH M,GONG Q,et al.Angiotensin II type 2 receptor blockade inhibits fatty acid synthase production through activation of AMP-activated protein kinase in pancreatic cancer cells[J].Surgery,2011,150(2):284-298.DOI:10.1016/j.surg.2011.06.002.
[10] VIAU A,BIENAIMé F,LUKAS K,et al.Cilia-localized LKB1 regulates chemokine signaling,macrophage recruitment,and tissue homeostasis in the kidney[J].EMBO J,2018,37(15):e98615.DOI:10.15252/embj.20179 8615.
[11] CLAUDE-TAUPIN A,FONDERFLICK L,GAUTHIER T,et al.ATG9A is overexpressed in triple negative breast cancer and its in vitro extinction leads to the inhibition of pro-cancer phenotypes[J].Cell,2018,7(12):E248.DOI:10.3390/cells7120248.
[12] FORRESTER A,DE LEONIBUS C,GRUMATI P,et al.A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex[J].EMBO J,2019,38(2):e99847.DOI:10.15252/embj.201899847.
[13] DING F,HU Q,WANG M,et al.Knockout of SlSBPASE suppresses carbon assimilation and alters nitrogen metabolism in tomato plants[J].Int J Mol Sci,2018,19(12):E4046.DOI:10.3390/ijms19124046.
[14] RAMANAGOUDR-BHOJAPPA R,CARRINGTON B,RAMASWAMI M,et al.Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth,sexual development and fertility[J].PLoS Genet,2018,14(12):e1007821.DOI:10.1371/journal.pgen.1007821.
[15] 刘菁,梁森,袁志.CRISPR/Cas9技术敲除faf1基因对斑马鱼软骨及肌节发育的影响[J].第三军医大学学报,2017,39(17):1709-1714.DOI:10.16.16/j.10000-5404.201612182.LIU J,LIANG S,YUAN Z.Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development[J].J Third Mil Med Univ,2017,39(17):1709-1714.DOI:10.16.16/j.10000-5404.201612182.
[16] KUANG X,LIU C,FANG J,et al.The tumor suppressor gene lkb1 is essential for glucose homeostasis during zebrafish early development[J].FEBS Lett,2016,590(14):2076-2085.DOI:10.1002/1873-3468.12237.
[17] ZHANG F,WEN Y,GUO X.CRISPR/Cas9 for genome editing:progress,implications and challenges[J].Hum Mol Genet,2014,23(R1):R40-R46.DOI:10.1093/hmg/ddu125.
[18] NGUYEN H B,BABCOCK J T,WELLS C D,et al.LKB1 tumor suppressor regulates AMP kinase/mTOR-independent cell growth and proliferation via the phosphorylation of Yap[J].Oncogene,2013,32(35):4100-4109.DOI:10.1038/onc.2012.431.
[19] LI J,LIU J,LI P P,et al.Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion[J].J Exp Clin Cancer Res,2014,33:70.DOI:10.1186/s13046-014-0070-0.
[20] SUN A,LI C,CHEN R,et al.GSK-3β controls autophagy by modulating LKB1-AMPK pathway in prostate cancer cells[J].Prostate,2016,76(2):172-183.DOI:10.1002/pros.23106.
[21] MARTIN S G,ST JOHNSTON D.A role for drosophila LKB1 in anterior-posterior axis formation and epithelial polarity[J].Nature,2003,421(6921):379-384.DOI:10.1038/nature01296.
[22] SHELLY M,POO M M.Role of LKB1-SAD/MARK pathway in neuronal polarization[J].Dev Neurobiol,2011,71(6):508-527.DOI:10.1002/dneu.20884.
[23] KWON S K,SANDO R,LEWIS T L,et al.LKB1 regulates mitochondria-dependent presynaptic calcium clearance and neurotransmitter release properties at excitatory synapses along cortical axons[J].PLoS Biol,2016,14(7):e1002516.DOI:10.1371/journal.pbio.1002516.
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