构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究

doi:10.3969/j.issn.1674-8115.2021.03.002

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (3): 297-301.doi: 10.3969/j.issn.1674-8115.2021.03.002

构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究

赵艳娜¹(), 邱荣²(), 沈南¹, 唐元家¹()

^1.上海交通大学医学院附属仁济医院风湿病科，上海市风湿病学研究所，上海 200127
^2.中国科学院上海营养与健康研究所，上海 200031

收稿日期:2020-03-11 出版日期:2021-03-28 发布日期:2021-04-06
通讯作者: 唐元家 E-mail:nanalr0704@163.com;rqiu@sibs.ac.cn;yjtang@sibs.ac.cn
作者简介:赵艳娜（1997—），女，硕士生；电子信箱：nanalr0704@163.com|邱荣（1989—），男，博士生；电子信箱：rqiu@sibs.ac.cn。
基金资助:
国家自然科学基金(81871287);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180100)

Construction of inducible CRISPR/Cas9 system for studying gene function in mouse

Yan-na ZHAO¹(), Rong QIU²(), Nan SHEN¹, Yuan-jia TANG¹()

^1.Shanghai Institute of Rheumatology, Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China

Received:2020-03-11 Online:2021-03-28 Published:2021-04-06
Contact: Yuan-jia TANG E-mail:nanalr0704@163.com;rqiu@sibs.ac.cn;yjtang@sibs.ac.cn
Supported by:
National Natural Science Foundation of China(81871287);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

摘要/Abstract

摘要：

目的·结合Dox诱导型单链导向RNA（single guide RNA，sgRNA）表达载体和Cas9转基因小鼠，构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究。方法·根据四环素诱导表达系统原理，基因合成U6-TetO-sgRNA和EF1α-T2A-Puro-BFP-T2A-TetR片段。通过同源重组将2个片段组装进反转录病毒载体骨架，获得Dox诱导型sgRNA反转录病毒载体。为了验证系统有效性，分离Cas9转基因小鼠骨髓细胞并诱导其向巨噬细胞方向分化。设计对照（non-targeting control，NC）组和实验组（靶向F4/80）的sgRNA，利用反转录病毒感染细胞，分化条件设置添加Dox组（Dox⁺）和不添加Dox组（Dox^-）。通过流式细胞术和T7核酸内切酶Ⅰ（T7 endonuclease Ⅰ，T7EⅠ）实验检测基因敲除效果。结果·①成功构建Dox诱导型sgRNA反转录病毒表达载体和Cas9转基因小鼠。②流式结果显示，在NC Dox^-组、NC^{Dox⁺组和F4/80 Dox^-组中，几乎无F4/80阴性细胞群体；而在F4/80Dox⁺组中，F4/80阴性细胞群体高达50%。③T7EⅠ结果显示，在F4/80 Dox^-组中，DNA条带完整，而在F4/80 Dox⁺组中发生基因突变，DNA条带被切开。结论·结合Dox诱导型sgRNA表达载体和Cas9转基因小鼠，成功构建诱导型CRISPR/Cas9系统。利用该系统成功在小鼠免疫细胞中实现可诱导性基因敲除。}

关键词: 诱导型CRISPR/Cas9系统, Dox 诱导型sgRNA表达载体, 造血干细胞, 基因编辑, 巨噬细胞

Abstract:

Objective·To construct inducible CRISPR/Cas9 system for studying gene function in mouse immune cells, combining Dox-inducible single guide RNA (sgRNA) expression vector with Cas9 transgenic mice.

Methods·U6-TetO-sgRNA and EF1α-T2A-Puro-BFP-T2A-TetR fragments were obtained by gene synthesis. The two synthetic fragments were assembled into the retroviral vector backbone by using homologous recombination. sgRNA targeting protein coding region of F4/80 and non-targeting control (NC) were designed. Bone marrow cells were isolated from Cas9 transgenic mice and transfected with retrovirus expressing sgRNA. The experimental conditions were divided into Dox-added group (Dox ⁺) and non Dox-added group (Dox^-). The knockout effect was tested by flow cytometry and T7 endonuclease Ⅰ （T7EⅠ) experiments.

Results·①Dox-inducible sgRNA retroviral vector and Cas9 transgenic mice were successfully constructed. ② The result of flow cytometry showed that F4/80 was only knocked out in the F4/80Dox⁺population, but not in NC Dox^-, NC Dox⁺ and F4/80 Dox^-populations. ③ T7EⅠ results showed that the DNA was cut into two bands in the F4/80Dox⁺ group, while the DNA band was intact in the F4/80 Dox^- group.

Conclusion·An inducible CRISPR/Cas9 system combining Dox-inducible sgRNA retroviral vector with Cas9 transgenic mice are successfully constructed. With this system, inducible gene knockout in mouse immune cells are successfully achieved.

Key words: inducible CRISPR/Cas9 system, Dox-inducible sgRNA expression vector, hematopoietic stem cell, gene editing, macrophage

中图分类号:

R394.4

赵艳娜, 邱荣, 沈南, 唐元家. 构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究[J]. 上海交通大学学报(医学版), 2021, 41(3): 297-301.

Yan-na ZHAO, Rong QIU, Nan SHEN, Yuan-jia TANG. Construction of inducible CRISPR/Cas9 system for studying gene function in mouse[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 297-301.

图/表 5

图1 Dox诱导型sgRNA表达载体构建策略Note: A. Components of inducible sgRNA retrovirus expression vector. B. Schematic diagram of Dox-inducible sgRNA expression.

Fig 1 Construction strategy of Dox-inducible sgRNA retroviral vector

图2 反转录病毒包装和NIH3T3细胞感染Note: A. Plat-E cells were transfected with the plasmid for 24 h, and the cell status was observed under white light by the microscope (×10). B. Plat-E cells were transfected with the plasmid for 48 h, and the expression of BFP was observed under the fluorescent microscope (×10). C. The retrovirus infection efficiency of NIH3T3 was detected by flow cytometry. BFP positive cells represent retrovirus-infected cells.

Fig 2 Retrovirus packaging and NIH3T3 cell infection

图3 流式细胞术检测骨髓来源的巨噬细胞中F4/80 表达Note: A. The expression of F4/80 was detected by flow cytometry. B. F4/80 expression was detected in the four experimental groups. ①P=0.000.

Fig 3 Flow cytometry detection of F4 / 80 expression in bone marrow-derived macrophage

图4 T7EⅠ实验检测F4/80基因组DNA编辑效率Note: Line 1 and 2 are the electrophoresis results of T7EⅠ experiment products in the F4/80 Dox- group; line 3 and 4 were the electrophoresis results of T7EⅠ experiment products in the F4/80 Dox+ group.

Fig 4 Detect DNA mutations of F4/80 locus by T7EⅠ

图5 反转录病毒感染小鼠骨髓造血干细胞Note: BFP-positive cells represent retrovirus-infected cells.

Fig 5 Retrovirus infection of mouse hematopoietic stem cells

参考文献 22

1	Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821.
2	Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol, 2011, 9(6): 467-477.
3	Makarova KS, Grishin NV, Shabalina SA, et al. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action[J]. Biol Direct, 2006, 1(1): 1-26.
4	Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Ⅲ[J]. Nature, 2011, 471(7340): 602-607.
5	Davis AJ,Chen DJ. DNA double strand break repair via non-homologous end-joining[J]. Transl Cancer Res, 2013, 2(3):130-143.
6	Butt H, Eid A, Ali Z, et al. Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide RNA molecule[J]. Front Plant Sci, 2017, 8: 1441.
7	Liang PP, Xu YW, Zhang XY, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes[J]. Protein Cell, 2015, 6(5): 363-372.
8	Pellagatti A, Dolatshad H, Valletta S, et al. Application of CRISPR/Cas9 genome editing to the study and treatment of disease[J]. Arch Toxicol, 2015, 89(7): 1023-1034.
9	Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J]. Cell, 2014, 159(2): 440-455.
10	Yin WX, Xiang P, Li QL. Investigations of the effect of DNA size in transient transfection assay using dual luciferase system[J]. Anal Biochem, 2005, 346(2): 289-294.
11	Adli M. The CRISPR tool kit for genome editing and beyond[J]. Nat Commun, 2018, 9(1): 1911.
12	Zhang XH, Tee LY, Wang XG, et al. Off-target effects in CRISPR/Cas9-mediated genome engineering[J]. Mol Ther Nucleic Acids, 2015, 4: e264.
13	Lin YN, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences[J]. Nucleic Acids Res, 2014, 42(11): 7473-7485.
14	Aubrey BJ, Kelly GL, Kueh AJ, et al. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo[J]. Cell Rep, 2015, 10(8): 1422-1432.
15	Cao J, Wu LZ, Zhang SM, et al. An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting[J]. Nucleic Acids Res, 2016, 44(19): e149.
16	Das AT, Tenenbaum L, Berkhout B. Tet-on systems for doxycycline-inducible gene expression[J]. Curr Gene Ther, 2016, 16(3): 156-167.
17	Herold MJ, van den Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of an shRNA-encoding Lentivirus in transgenic rats[J]. PNAS, 2008, 105(47): 18507-18512.
18	Polstein LR, Gersbach CA. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation[J].Nat Chem Biol, 2015, 11(3):198-200.
19	Dow LE, Fisher J, O'Rourke KP, et al. Inducible in vivo genome editing with CRISPR-Cas9[J]. Nat Biotechnol, 2015, 33(4): 390-394.
20	Lu J, Zhao C, Zhao YZ, et al. Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing[J]. Nucleic Acids Res, 2018, 46(5): e25.
21	de Solis CA, Ho A, Holehonnur R, et al. The development of a viral mediated CRISPR/Cas9 system with doxycycline dependent gRNA expression for inducible in vitro and in vivo genome editing[J]. Front Mol Neurosci, 2016, 9: 70.
22	LaFleur MW, Nguyen TH, Coxe MA, et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system[J]. Nat Commun, 2019, 10: 1668.

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构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究

Construction of inducible CRISPR/Cas9 system for studying gene function in mouse

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