CRISPR/Cas9介导下高效敲除SQSTM1/p62基因的Hela细胞模型的建立
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摘要
目的 :探讨针对p62双sgRNA向导CRISPR/Cas9基因编辑效率。方法 :采用CRISPR/Cas9基因编辑技术成功敲除p62基因,通过细胞有限稀释法和嘌呤霉素筛选,免疫印迹法验证双sgRNA和单sgRNA向导的基因编辑成功率;流式细胞仪验证p62缺失对Hela细胞凋亡的影响。结果 :免疫印迹分析得出双sgRNA导向的p62敲除效率高于单sgRNA导向的敲除,靶序列测序比对分析确认p62编码基因发生大片段缺失突变。H_2O_2处理稳定敲除的Hela细胞系显示,p62基因敲除能明显抑制Hela细胞H_2O_2诱导的早期细胞凋亡。结论 :成功建立了p62敲除的Hela细胞系,双sgRNA向导的CRISPR/Cas9基因编辑体系可能是一种更有效的编辑工具。
Objective : To explore the editing efficiency of the dual sgRNA guided CRISPR/Cas9 gene targeting p62 protein. Methods : Hela cells with p62 knockout were screened with limited dilution and puromycin treatment after CRISPR/Cas9 gene editing, the success rate of gene editing guided by dual sgRNA and single sgRNA was verified using Western blotting, and the rate of apoptosis was analyzed by flow cytometry. Results : Western blotting analysis indicated that the effect of knockout via dual sgRNA-targeted p62 was elevated than that of single sgRNA, and targetsequence comparison revealed that the large fragment was deleted in p62 knockout cells. After H_2O_2 treatment, the p62 knockout cells inhibited early apoptosis in Hela cells. Conclusion : The p62 knockout Hela cell line is successfully established, and the CRISPR/Cas9 gene editing system guided by dual sgRNA may be a more effective editing tool.
Objective : To explore the editing efficiency of the dual sgRNA guided CRISPR/Cas9 gene targeting p62 protein. Methods : Hela cells with p62 knockout were screened with limited dilution and puromycin treatment after CRISPR/Cas9 gene editing, the success rate of gene editing guided by dual sgRNA and single sgRNA was verified using Western blotting, and the rate of apoptosis was analyzed by flow cytometry. Results : Western blotting analysis indicated that the effect of knockout via dual sgRNA-targeted p62 was elevated than that of single sgRNA, and targetsequence comparison revealed that the large fragment was deleted in p62 knockout cells. After H_2O_2 treatment, the p62 knockout cells inhibited early apoptosis in Hela cells. Conclusion : The p62 knockout Hela cell line is successfully established, and the CRISPR/Cas9 gene editing system guided by dual sgRNA may be a more effective editing tool.
引文
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[2]Komatsu M,Kageyama S,Ichimura Y.p62/SQSTM1/A170:physiology and pathology[J].Pharmacol Res,2012,66(6):457-462.
[3]Barrangou R,Fremaux C,Deveau H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.
[4]Kaboli S,Babazada H.CRISPR mediated genome engineering and its application in industry[J].Curr Issues Mol Biol,2018,26:81-92.
[5]Torres Ruiz R,Rodriguez Perales S.CRISPR-Cas9:a revolutionary tool for cancer modelling[J].Int J Mol Sci,2015,16(9):22151-22168.
[6]Horii T,Hatada I.Genome editing using mammalian haploid cells[J].Int J Mol Sci,2015,16(10):23604-23614.
[7]Plaza Reyes A,Lanner F.Towards a CRISPR view of early human development:applications,limitations and ethical concerns of genome editing in human embryos[J].Development,2017,144(1):3-7.
[8]Takayama K,Igai K,Hagihara Y,et al.Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system[J].Nucleic Acids Res,2017,45(9):5198-5207.
[9]Liang X,Potter J,Kumar S,et al.Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA,Cas9 nuclease,and donor DNA[J].J Biotechnol.2017,241:136-146
[10]Cohen Kaplan V,Livneh I,Avni N,et al.p62-and ubiquitindependent stress-induced autophagy of the mammalian 26Sproteasome[J].Proc Natl Acad USA,2016,113(47):E7490-E7499
[11]Hou W,Han J,Lu C,et al.Autophagic degradation of active caspase-8:a crosstalk mechanism between autophagy and apoptosis[J].Autophagy,2010,6(7):891-900.
[12]俞晨,张俊强,储海峰,等.白藜芦醇对过氧化氢诱导滑膜细胞凋亡的作用及机制[J].解剖学杂志,2016,39(4):431-435.
[13]Moscat J,Diaz-Meco MT.p62:a versatile multitasker takes on cancer[J].Trends Biochem Sci,2012,37(6):230-236.
[14]Ichimura Y,Komatsu M.Activation of p62/SQSTM1-Keap1-nuclear factor erythroid 2-related factor 2 pathway in cancer[J].Front Oncol,2018,8:210.
[15]Miller L,Rollinson S,Callister JB,et al.p62/SQSTM1 analysis in frontotemporal lobar degeneration[J].Neurobiol Aging,2015,36(3):1603.e5-9.
[16]Saito T,Ichimura Y,Taguchi K,et al.p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming[J].Nat Commun,2016,7:12030.
[17]Bitto A,Lerner CA,Nacarelli T,et al.P62/SQSTM1 at the interface of aging,autophagy,and disease[J].Age(Dordr),2014,36(3):9626.
[18]Zach F,Polzer F,Mueller A,et al.p62/sequestosome 1 deficiency accelerates osteoclastogenesis in vitro and leads to Paget's diseaselike bone phenotypes in mice[J].J Biol Chem,2018,293(24):9530-9541.
[19]Xu Y,Zhang J,Tian C,et al.Overexpression of p62/SQSTM1promotes the degradations of abnormally accumulated PrPmutants in cytoplasm and relieves the associated cytotoxicities via autophagy-lysosome-dependent way[J].Med Microbiol Immunol,2014,203(2):73-84.
[20]Cermak T,Doyle EL,Christian M,et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J].Nucleic Acids Res,2011,39(12):e82.
[21]Lone BA,Karna SKL,Ahmad F,et al.CRISPR/Cas9 system:a bacterial tailor for genomic engineering[J].Genet Res Int,2018,2018:3797214
[22]Mali P,Yang L,Esvelt KM,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.