建立靶向CXCR7基因的CRISPR/Cas9 基因编辑系统及其应用

doi:10.3969/j.issn.1000-8535.2020.05.004

广州医药 ›› 2020, Vol. 51 ›› Issue (5): 16-20.DOI: 10.3969/j.issn.1000-8535.2020.05.004

建立靶向CXCR7基因的CRISPR/Cas9 基因编辑系统及其应用

吴洁莹^1,2, 孙逊沙³, 李发涛^1,2, 陈劲松^1,2, 谢闺娥⁴, 李焱^1,2, 吴韶清^1,2

1 广州医科大学附属广州市妇女儿童医疗中心(广州 510623)
2 中山大学附属广州市妇女儿童医疗中心(广州 510623)
3 中山大学附属第一医院(广州 510080)
4 广州医科大学金域检验学院(广州 510182)

收稿日期:2020-04-24 出版日期:2020-09-20 发布日期:2021-11-28
通讯作者: 吴韶清,E-mail:wushaoqing2005@21cn.com
基金资助:
国家自然科学基金项目(81601004,81102248);广东省自然科学基金(2016A030310139,2017A030313906);广东省科技计划项目(2014A020212019)

Establishment and application of CRISPR/Cas9 gene editing system targeting CXCR7 gene

WU Jieying^1,2, SUN Xunsha³, LI Fatao^1,2, CHEN Jinsong^1,2, XIE Guie⁴, LI Yan^1,2, WU Shaoqing^1,2

1 Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China
2 Guangzhou Women and Children's Medical Center, Sun Yat-sen University, Guangzhou 510623, China
3 The First Affiliated Hospital,Sun Yat-sen University, Guangzhou 510080, China
4 KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510182,China

Received:2020-04-24 Online:2020-09-20 Published:2021-11-28

摘要/Abstract

摘要： 目的构建靶向CXCR7基因的CRISPR/Cas9基因编辑系统,并应用于HEK 293T细胞系。方法设计两对靶向CXCR7基因的sgRNAs,分别插入PX458载体中,并转化DH5α大肠埃希菌。经菌液PCR和测序验证,挑选序列正确的sgRNA-CXCR7-PX458质粒,转染HEK 293T细胞,用流式分选转染阳性细胞,提取其DNA,PCR扩增后测序验证。结果经测序验证,成功构建了靶向CXCR7基因的CRISPR/Cas9系统,转染HEK 293T细胞后,测序鉴定发现成功编辑CXCR7基因。结论成功构建了靶向CXCR7的sgRNA-CXCR7-PX458质粒,可在HEK 293T上成功编辑CXCR7基因,为进一步的功能研究奠定基础。

关键词: CRISPR/Cas9技术, 基因编辑, CXCR7

Abstract: Objective To construct the CRISPR/Cas9 gene editing system targeting C-X-C chemokine receptor 7 (CXCR7) gene and to edit CXCR7 gene in 293T cell line. Methods Two pairs of small guide RNAs (sgRNAs) targeting CXCR7 gene were designed and inserted into PX458 vector, which were transformed into host bacterium Escherichia coliDH5α. The correct sgRNA-CXCR7-PX458 plasmids were selected by PCR and further Sanger sequencing verification. HEK 293T cell line was transfected by DNA of sgRNA-CXCR7-PX458 plasmid. After 72 hours,GFP-positive cells were sorted by flow cytometry. We did DNA extraction of the GFP-positive cells and amplified the CXCR7 gene corresponding fragment by PCR and investigated the CXCR7 gene editing results by Sanger sequencing. Results The CRISPR/Cas9 system targeting CXCR7 gene was successfully constructed. After 293T cells were transfected, the CXCR7 gene was edited in HEK 293T cells successfully. Conclusion The sgRNA-CXCR7-PX458 plasmid targeting CXCR7 gene was successfully constructed. The CRISPR/Cas9 gene editing system targeting CXCR7 gene were used on the HEK 293T cell line, which lays a foundation for further study of BCOR function.

Key words: CRISPR/Cas9 technology, Gene editing, CXCR7

吴洁莹, 孙逊沙, 李发涛, 陈劲松, 谢闺娥, 李焱, 吴韶清. 建立靶向CXCR7基因的CRISPR/Cas9 基因编辑系统及其应用[J]. 广州医药, 2020, 51(5): 16-20.

WU Jieying, SUN Xunsha, LI Fatao, CHEN Jinsong, XIE Guie, LI Yan, WU Shaoqing. Establishment and application of CRISPR/Cas9 gene editing system targeting CXCR7 gene[J]. Guangzhou Medical Journal, 2020, 51(5): 16-20.

参考文献

[1] RAN F A, HSU P D, WRIGHT J, et al. Genome engineering using the CRISPR-Cas9 system[J]. Nat Protoc, 2013, 8(11):2281.
[2] NAKAZAWA T, NATSUME A, NISHIMURA F, et al. Effect of CRISPR/Cas9-mediated PD-1-disrupted primary human third-generation CAR-T cells targeting EGFRvIII on In vitro human glioblastoma cell growth[J]. Cells, 2020, 9(4):998.
[3] ZHANG H X, ZHANG Y, YIN H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9 [J]. Mol Ther, 2019, 27(4):735-746.
[4] MA Y, ZHANG L, HUANG X.Genome modification by CRISPR/Cas9 [J]. FEBS J, 2014, 281(23):5186-93.
[5] MOJICA FJM, MONTOLIU L. On the origin of CRISPR-cas technology: from prokaryotes to mammals [J]. Trends Microbiol, 2016, 24(10):811-820.
[6] XU L, WANG J, LIU Y, et al. CRISPR-Edited stem cells in a patient with HIV and acute lymphocytic leukemia [J]. N Engl J Med, 2019, 381(13):1240-1247.
[7] TASCA F, WANG Q, GONÇALVES M. Adenoviral vectors meet gene editing: a rising partnership for the genomic engineering of human stem cells and their progeny [J]. Cells, 2020, 9(4):953.
[8] VALENTI M T, SERENA M, CARBONARE L D, et al. CRISPR/Cas system: an emerging technology in stem cell research [J]. World J Stem Cell, 2019, 11(11):937-956.
[9] NEGORO R, KAWAI K, ICHIKAWA M, et al. Establishment of MDR1-knockout human induced pluripotent stem cell line [J]. Drug Metab Pharmacokinet, 2020(1):9.
[10] WANG H, WANG M, WANG Y, et al. MSX2 suppression through inhibition of TGFβ signaling enhances hematopoietic differentiation of human embryonic stem cells [J]. Stem Cell Res Ther, 2020, 11(1):147.
[11] GOLCHIN A, SHAMS F, KARAMI F. Advancing mesenchymal stem cell therapy with CRISPR/cas 9 for clinical trial studies [J]. Adv Exp Med Biol. 2020(1):459.
[12] KIM Y K, YU J H, MIN S H, et al. Generation of a GLA knock-out human-induced pluripotent stem cell line, KSBCi002-A-1, using CRISPR/Cas9 [J]. Stem Cell Res, 2020(42):101676.
[13] BALABANIAN K, LAGANE B, INFANTINO S, et al. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes [J]. J Biol Chem, 2005, 280(42):35760-35766.
[14] TARNOWSKI M, LIU R, WYSOCZYNSKI M, et al.CXCR7: a new SDF-1-binding receptor in contrast to normal CD34(+) progenitors is functional and is expressed at higher level in human malignant hematopoietic cells [J].Eur J Haematol, 2010, 85(6):472-483.
[15] WANG C, CHEN W, SHEN J. CXCR7 targeting and its major disease relevance [J]. Front Pharmacol, 2018(9):641.
[16] SÁNCHEZ-MARTÍN L, SÁNCHEZ-MATEOS P, CABAÑAS C. CXCR7 impact on CXCL12 biology and disease [J]. Trends Mol Med, 2013, 19(1):12-22.
[17] KOWALSKI K, BRZOSKA E, CIEMERYCH M A. The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation [J]. Stem Cell Res, 2019(41):101636.
[18] SHAO Y, ZHOU F, HE D, et al. Overexpression of CXCR7 promotes mesenchymal stemcells to repair phosgene-induced acute lung injury in rats [J]. Biomed Pharmacother, 2019, 109 (1):1233-1239.
[19] CHEN Q, ZHANG M, LI Y,et al. CXCR7 mediates neural progenitor cells migration to CXCL12 independent of CXCR4 [J]. Stem Cells, 2015, 33(8):2574-2585.

建立靶向CXCR7基因的CRISPR/Cas9 基因编辑系统及其应用

Establishment and application of CRISPR/Cas9 gene editing system targeting CXCR7 gene

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