利用CRISPR/Cas9技术建立敲除JAK2基因K562细胞系
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摘要
目的:利用CRISPR/Cas9技术对K562细胞系JAK2基因进行编辑,构建JAK2基因敲除的K562细胞系。方法:使用CRISPR在线设计工具,针对JAK2基因设计sgRNA,构建Cas9-sgRNA共表达质粒。使用第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,Sanger测序和TA克隆检测基因编辑活性。无限稀释法将编辑阳性的细胞接种于96孔板并扩培得到单克隆细胞株,提取基因组DNA,Sanger测序和TA克隆分析敲除JAK2单克隆细胞的基因型。结果:成功构建靶向敲除JAK2基因的lentiCRISPRv2-sgRNA3-1质粒。优化方案得到低细胞毒性高转染效率的感染K562细胞慢病毒量。CRISPR/Cas9系统成功在JAK2基因sgRNA3-1识别位点发挥基因组编辑活性,获得纯合敲除JAK2基因细胞株K562-JAK2~(-/-)(两个等位分别发生移码突变,预期编码没有功能的JAK2蛋白)。结论:CRIAPR/Cas9系统通过慢病毒感染方式获得JAK2基因纯合敲除的K562细胞株,该细胞模型可用于研究在慢性髓系白血病中JAK2基因的作用,为构建K562敲除其他基因细胞系提供实验依据,为探究造血分化机制的研究奠定实验基础。
Objective: To generate a JAK2 knockout K562 cell line by CRISPR/Cas9 gene editing system.Methods: Using CRISPR online design tool,sgRNA was designed targeted to JAK2 gene and used to construct the co-expression plasmid of Cas9-sgRNA. Lentivirus was packaged by the second-generation lentivirus packaging system and used to transduce K562 cells,the genomic DNA of cell pool was extracted,and the gene editing activity was detected by Sanger sequencing and TA cloning. The candidate edited cells were inoculated into 96-well plate by infinite dilution method. Then the cells were expanded to extract genomic DNA. The JAK2 sequence was identified by Sanger sequencing and TA cloning. Results: LentiCRISPRv2-sgRNA3-1 plasmid containing the gene editing tools for koncking out JAK2 was constructed. The optimal dose of lentivirus with low cytotoxicity and high transduction efficiency was obtained. The JAK2 gene knockout K562 cell line(K562-JAK2~(-/-)) was successfully generated. Conclusion: Lentivirus transduction-based CRISPR/Cas9 system was successfully used to generate JAK2 gene homozygous knockout K562 cell line,providing a cell model to study the importance of JAK2 gene in the field of chronic myeloid leukemia. Besides,the lentivirus transduction-based CRISPR/Cas9 protocol reported here lays a foundation for constructing other gene knockout K562 cell lines to study of hematopoietic differentiation mechanism.
Objective: To generate a JAK2 knockout K562 cell line by CRISPR/Cas9 gene editing system.Methods: Using CRISPR online design tool,sgRNA was designed targeted to JAK2 gene and used to construct the co-expression plasmid of Cas9-sgRNA. Lentivirus was packaged by the second-generation lentivirus packaging system and used to transduce K562 cells,the genomic DNA of cell pool was extracted,and the gene editing activity was detected by Sanger sequencing and TA cloning. The candidate edited cells were inoculated into 96-well plate by infinite dilution method. Then the cells were expanded to extract genomic DNA. The JAK2 sequence was identified by Sanger sequencing and TA cloning. Results: LentiCRISPRv2-sgRNA3-1 plasmid containing the gene editing tools for koncking out JAK2 was constructed. The optimal dose of lentivirus with low cytotoxicity and high transduction efficiency was obtained. The JAK2 gene knockout K562 cell line(K562-JAK2~(-/-)) was successfully generated. Conclusion: Lentivirus transduction-based CRISPR/Cas9 system was successfully used to generate JAK2 gene homozygous knockout K562 cell line,providing a cell model to study the importance of JAK2 gene in the field of chronic myeloid leukemia. Besides,the lentivirus transduction-based CRISPR/Cas9 protocol reported here lays a foundation for constructing other gene knockout K562 cell lines to study of hematopoietic differentiation mechanism.
引文
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[20]Zetsche B,Gootenberg J S,Abudayyeh O O,et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell,2015,163(3):759-771.
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[22]Mali P,Aach J,Stranges P B,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nature Biotechnology,2013,31(9):833-838.
[23]Rees H A,Komor A C,Yeh W H,et al.Improving the DNAspecificity and applicability of base editing through protein engineering and protein delivery.Nature Communications,2017,8(1):15790.
[24]Rees H A,Liu D R.Base editing:precision chemistry on the genome and transcriptome of living cells.Nature Reviews Genetics,2018,19(1):770-788.
[25]Shao Y,Guan Y,Wang L,et al.CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos.Nature Protocols,2014,9(10):2493-2512.
[26]Song B,Fan Y,He W,et al.Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.Stem Cells and Development,2015,24(9):1053-1065.
[2]Chen M,Gallipoli P,Degeer D,et al.Targeting primitive chronic myeloid leukemia cells by effective inhibition of a new AHI-1-BCR-ABL-JAK2 complex.Journal of the National Cancer Institute,2013,105(6):405-423.
[3]Warsch W,Grundschober E,Berger A,et al.STAT5 triggers BCR-ABL1 mutation by mediating ROS production in chronic myeloid leukaemia.Oncotarget,2012,3(12):1669-1687.
[4]James C,Ugo V,Le Couédic J P,et al.A unique clonal JAK2mutation leading to constitutive signalling causes polycythaemia vera.Nature,2005,434(7037):1144-1148.
[5]Hartlerode A J,Scully R.Mechanisms of double-strand break repair in somatic mammalian cells.Biochemical Journal,2009,423(2):157-168.
[6]Chandrasegaran S.Recent advances in the use of ZFN-mediated gene editing for human gene therapy.Cell&Gene Therapy Insights,2017,3(1):33-41.
[7]Zhang Z,Wu W S.Application of TALE-Based approach for dissecting functional microRNA-302/367 in cellular reprogramming.Methods Mol Biol,2018,1733:255-263.
[8]Jinek M,Chylinski K,Fonfara I,et al.A programmable Dual-RNA-Guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[9]Ran Fa,Hsu P D,Wright J,et al.Genome engineering using the CRISPR-Cas9 system.Nature Protocols,2013,8(11):2281-2308.
[10]Shen B.Generation of gene-modified mice via cas9/RNA-mediated gene targeting.Cell Research,2013,23(5):720-723.
[11]Niu Y,Shen B,Cui Y,et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.Cell,2014,156(4):836-843.
[12]Sánchez-Rivera F J,Papagiannakopoulos T,Romero R,et al.Rapid modelling of cooperating genetic events in cancer through somatic genome editing.Nature,2014,516(7531):428-431.
[13]Liao H K,Gu Y,Diaz A,et al.Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.Nature Communications,2015,6(1):6413.
[14]Kotterman M A,Schaffer D V.Engineering adeno-associated viruses for clinical gene therapy.Nature Reviews Genetics,2014,15(7):445-451.
[15]Xu L,Yang H,Gao Y,et al.CRISPR/Cas9-Mediated CCR5ablation in Human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo.Molecular Therapy,2017,25(8):1782.
[16]Neviani P,Harb J G,Oaks J J,et al.PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells.Journal of Clinical Investigation,2013,123(10):4144-4157.
[17]Warsch W,Grundschober E,Sexl V.Adding a new facet to STAT5in CML:multitasking for leukemic cells.Cell Cycle,2013,12(12):2.
[18]Weber A,Borghouts C,Brendel C,et al.Stat5 exerts distinct,vital functions in the cytoplasm and nucleus of Bcr-Abl+K562 and Jak2(V617F)+HEL leukemia cells.Cancers,2015,7(1):503.
[19]Gallipoli P,Cook A,Rhodes S,et al.JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of chronic phase CML CD34+cells in vitro and in vivo.Blood,2014,124(9):1492-501.
[20]Zetsche B,Gootenberg J S,Abudayyeh O O,et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell,2015,163(3):759-771.
[21]Abudayyeh O O,Gootenberg J S,Essletzbichler P,et al.RNAtargeting with CRISPR-Cas13.Nature,2017,550(7675):280-284.
[22]Mali P,Aach J,Stranges P B,et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nature Biotechnology,2013,31(9):833-838.
[23]Rees H A,Komor A C,Yeh W H,et al.Improving the DNAspecificity and applicability of base editing through protein engineering and protein delivery.Nature Communications,2017,8(1):15790.
[24]Rees H A,Liu D R.Base editing:precision chemistry on the genome and transcriptome of living cells.Nature Reviews Genetics,2018,19(1):770-788.
[25]Shao Y,Guan Y,Wang L,et al.CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos.Nature Protocols,2014,9(10):2493-2512.
[26]Song B,Fan Y,He W,et al.Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.Stem Cells and Development,2015,24(9):1053-1065.