Establishment of CSE1L Knockout C2C12 Cells by CRISPR-Cas9 System

doi:10.13304/j.nykjdb.2024.0250

Abstract

Abstract:

To study the role of chromosomeseegregation 1-like （CSE1L） protein in skeletal muscle development in mice， CSE1L gene knockout C2C12 cell line was constructed by CRISPR-Cas9 gene editing technology. 3 pairs of single RNAs （sgRNAs） targeting the third exon of mouse CSE1L CDS sequence were designed， then sgRNA plasmid （linked to green fluorescent protein， GFP） and Cas9 plasmid （linked to red fluorescent protein， RFP） were constructed. The sgRNA and Cas9 were co-transfected into C2C12 cell line， then the cleavage efficiency of different sgRNA was screened. After co-transfection， the C2C12 cell lines were sorted by flow cytometry to obtain the cells co-expressing GFP and RFP， and then identified by DNA sequencing. The molecular characteristics of CSE1L were identified by real-time quantitative PCR （RT-qPCR） and Western blot. Finally， CCK-8， cell cycle and apoptosis test were used to examine the activity and apoptosis of CSE1L knock out cell line. The results showed that CSE1L gene mRNA and protein expression level were significantly decreased （P<0.01）， which indicated that CSE1L gene was successfully knocked out. CSE1L gene deletion resulted in C2C12 cell line elongated， cell viability was significantly down-regulated at 72 h， cell cycle arrest and apoptosis increased. This study demonstrated the CSE1L gene knockout cell line was successfully constructed， and the function related to growth and development of mouse skeletal muscle cells was studied， which provided a cell line model for exploring the function of CSE1L gene study and laid a foundation for generating subsequent gene editing knockout mice.

Key words: CRISPR/Cas9, CSE1L, gene knockout, skeletal muscle development

摘要：

为研究染色体分离样蛋白1（chromosomesegregation 1-like，CSE1L）在小鼠骨骼肌发育中的作用，利用CRISPR-Cas9基因编辑技术构建CSE1L基因敲除的C2C12细胞系。针对小鼠 CSE1L的第3外显子设计3组sgRNA，构建sgRNA表达质粒（连接绿色荧光蛋白，green fluorescent protein，GFP）和Cas9质粒（连接红色荧光蛋白，red fluorescent protein，RFP）共转染C2C12细胞，筛选高切割效率的sgRNA；将转染该sgRNA和Cas9的C2C12细胞进行流式细胞分选，获取共表达红、绿双色荧光蛋白的细胞，并用DNA测序进行鉴定。通过实时荧光定量PCR（RT-qPCR）和蛋白免疫印迹（Western-blot）鉴定其分子特征。最后分别利用CCK-8、细胞周期以及凋亡实验对CSE1L敲除细胞系活力和凋亡进行检测。结果显示，小鼠成肌细胞中CSE1L基因mRNA和蛋白水平表达量都显著降低（P<0.01），提示CSE1L基因敲除成功。该基因缺失导致C2C12细胞变细长、细胞活力在72 h显著下调，细胞周期停滞、凋亡增加。成功构建了CSE1L基因敲除的细胞系，并研究了小鼠骨骼肌细胞生长发育相关功能，为探索CSE1L基因功能提供细胞模型，并为后续基因编辑敲除鼠制备奠定基础。

关键词: CRISPR-Cas9, CSE1L, 基因敲除, 骨骼肌发育

CLC Number:

Zixin LI, Hongfei BAI, Yong XIE, Xun LI, Lijing BAI. Establishment of CSE1L Knockout C2C12 Cells by CRISPR-Cas9 System[J]. Journal of Agricultural Science and Technology, 2024, 26(11): 225-233.

李紫馨, 白鸿飞, 谢勇, 李珣, 白立景. 基于CRISPR-Cas9技术建立CSE1L基因敲除C2C12细胞[J]. 中国农业科技导报, 2024, 26(11): 225-233.

Figures/Tables 7

Table 1 Primer sequence

基因名称Gene name	序列Sequence（5’-3’）	用途Purpose
sgRNA-1	F：CACCGAAGTTTCTCGAATCCGTAGAR：AAACTCTACGGATTCGAGAAACTTC	载体构建 Vector construction
sgRNA-2	F：CACCGAATAATGTTAAAAGCAACAGR：AAACCTGTTGCTTTTAACATTATTC
sgRNA-3	F：CACCGAACATTATTGGAAAAGTCACR：AAACGTGACTTTTCCAATAATGTTC
CSE1L-1	F： GAAGACCACCTTGGACCTTTAA R： CCTGCTGTGAAATAAATGGACGAAT	PCR
CSE1L-2	F： GCGATGCGAATTTACAGACACT R： AAGCAACAGTGGGTAATTCTGA	RT-qPCR
Gapdh	F：AGGTCGGTGTGAACGGATTTG R： TGTAGACCATGTAGTTGAGGTCA	RT-qPCR

Fig. 1 DNA sequencing results of wild type and CSE1L-/- cellA：Flow cytometry of normal cells； B：Flow cytometry of CSE1L-/- cells. Q4 is the negative cells population， Q2 is the FITC-positive and PE-positive cells

Fig. 2 Comparison of cell morphology between wild type and CSE1L knockout cells

Fig. 3 RT-qPCR and Western blot detection of the mRNA and protein of CSE1L gene in wild type and CSE1L knockout cell lineA：mRNA level；B：Protein level；*** indicates significant difference at P<0.001 level.

Fig. 4 Effects of CSE1L knockout on cell viability of C2C12 cellsNote： * and ** indicate significant differences between treatments at P<0.01 and P<0.001 levels， respectively.

Fig. 5 Effects of CSE1L knockout on cell cycle of C2C12 cellsA： Flow cytometry cell cycle analysis of C2C12 and CSE1L-/- cells； B： Statistical results of flow cytometry. * indicates significant difference at P <0.05 level

Fig. 6 Effects of CSE1L knockout on C2C12 cell apoptosisA： Flow cytometry analysis of cell apoptosis； B： Radio of normal apoptotic cells at different stage. ** indicates significant difference at P<0.01 level

References 32

1	BEHRENS P, BRINKMANN U, WELLMANN A. CSE1L/CAS:Its role in proliferation and apoptosis [J]. Apoptosis,2003, 8(1): 39-44.
2	BEHRENS P, BRINKMANN U, FOGT F, et al.. Implication of the proliferation and apoptosis associated CSE1L/CAS gene for breast cancer development [J]. Anticancer Res., 2001,21(4A):2413-2417.
3	TANAKA T, OHKUBO S, TATSUNO I, et al.. hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes [J]. Cell, 2007, 130(4): 638-650.
4	刘小玉,李旭东,关坤萍.沉默CSE1L对急性髓系白血病细胞阿糖胞苷敏感性的影响及作用机制研究[J].中国病理生理杂志,2023,39(2): 269-275.
	LIU X Y, LI X D, GUAN K P. Effect of CSE1L silencing on cytarabine sensitivity of acute myeloid leu-kemia cells [J]. Chin. J. Pathophysiol., 2023, 39(2): 269-275.
5	王婧.CAS/CSE1L在急性白血病中的表达水平及对其临床意义的探讨[D].太原:山西医科大学,2022.
	WANG Q. Expression level of CAS/CSE1L in acute leukemia and its clinical significance [D]. Taiyuan: Shanxi Medical University, 2022.
6	DONG Q, LI X, WANG C Z,et al.. Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing [J]. Proc. Natl. Acad. Sci. USA, 2018,115(17):E4013-E4022.
7	徐宾.Circ_cse1l在结直肠癌中的表达及相关功能研究[D].石家庄:河北医科大学,2021.
	XU B. Expression and function syudy of Circ_cse1l in colorectal cancer [D]. Shijiazhuang: Hebei Medical Unibersity, 2021.
8	马晨博,杨能红. CSE1L在恶性肿瘤中的表达及治疗应用研究进展[J].现代医药卫生, 2019,35(5):698-701.
9	袁新.染色体分离1样基因(CSE1L)在肝癌中的表达及临床意义[D].郑州:郑州大学,2021.
	YUAN X. Expression and clinical significance of chromosome segregation 1-like in hepatocellular carcinoma [D]. Zhenzhou: Zhenzhou University, 2021.
10	LIU C Y, WEI J J, XU K, et al.. CSE1L participates in regulating cell mitosis in human seminoma [J/OL]. Cell Prolif., 2019, 52(2): e12549 [2024-07-19]. .
11	TENG J Y, GAO Y H, YIN H W, et al.. A compendium of genetic regulatory effects across pig tissues [J]. Nat. Genet., 2024, 56(1): 112-123.
12	WANG J Y, DOUDNA J A. CRISPR technology: a decade of genome editing is only the beginning [J/OL]. Science, 2023, 379(6629): eadd8643 [2024-07-19]. .
13	KWON D H, GIM G M, YUM S Y, et al..Current status and future of gene engineering in livestock [J]. BMB Rep., 2024, 57(1): 50-59.
14	PACESA M, PELEA O, Past JINEK M., present,and future of CRISPR genome editing technologies [J]. Cell, 2024, 187(5): 1076-1100.
15	谢春嫡,郭肖蓉,张煦栋,等.基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞[J].中国农业科技导报,2022,24(10):200-207.
	XIE C D, GUO X R, ZHANG X D, et al.. Establishment of IGF2R knockout PK-15 cells by CRISPR/Cas9 system [J]. J. Agric. Sci. Technol., 2022, 24(10): 200-207.
16	FRESHNEY R I. Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications [M].Sixth Edn. Hoboken, N. J.:Wiley-Blackwell, 2010: 1-732.
17	黄新磊.纳米紫杉醇共组装系统通过稳定微管细胞骨架减轻缺血性卒中神经元死亡的实验研究[D].扬州:扬州大学,2023.
	HUANG X L. Experimental study of nano-paclitaxel co-assembly system to alleviate neuronal death in ischemic stroke by stabilizing microtubule cytoskeleton [D]. Yangzhou:Yangzhou University, 2023.
18	周倩宇.细胞骨架微管蛋白在PHEV感染神经细胞过程中的作用及其机制[D].长春:吉林大学,2022.
	ZHUO Q Y.The function and mechanism of microtubule cytoskeleton in porcine hemagglutinating encephalomyelitis virus infecting nerve cells [D]. Changchun: Jilin University, 2022.
19	张继华,李浩,张媛媛,等. CSE1L在人喉鳞癌颈淋巴结转移中的作用研究[J].河北医科大学学报,2021,24(12):1417-1420, 1436.
	ZHANG J H, LI H, ZHANG Y Y, et al.. Role of CSE1L in cervical lymph node metastasis of human laryngeal squamous cell carcinoma[J]. J. Hebei Med. Univ., 2021,24(12):1417-1420, 1436.
20	LIAO C F, LUO S F, SHEN T Y, et al.. CSE1L/CAS, a microtubule-associated protein,inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs [J]. BMB Rep., 2008,41(3):210-216.
21	刘小玉.沉默CSE1L增强急性髓系白血病细胞对阿糖胞苷敏感性的机制研究[D].太原:山西医科大学,2023.
	LIU X Y. Mechanism of CSE1L silencing to enhance the sensitivity of acute myeloid leukemia cells to Cytarabine [D]. Taiyuan: Shanxi Medical University, 2023.
22	JIANG M C. CAS (CSE1L) signaling pathway in tumor progression and its potential as a biomarker and target for targeted therapy [J].Tumor. Biol., 2016,37(10):13077-13090.
23	WELLMANN A, FLEMMING P, BEHRENS P, et al..High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: correlation with tumor progression [J]. Int. J. Mol. Med, 2001,7(5):489-494.
24	SEIDEN-LONG I M, BROWN K R, SHIH W, et al.. Transcriptional targets of hepatocyte growth factor signaling and Ki-ras oncogene activation in colorectal cancer [J]. Oncogene, 2006, 25(1): 91-102.
25	WELLMANN A, KRENACS L, FEST T, et al.. Localization of the cell proliferation and apoptosis-associated CAS protein in lymphoid neoplasms [J]. Am. J. Pathol., 1997,150(1):25-30.
26	PEIRÓ G, DIEBOLD J, BARETTON G B, et al.. Cellular apoptosis susceptibility gene expression in endometrial carcinoma:correlation with Bcl-2,Bax,and caspase-3 expression and outcome [J]. Int. J. Gynecol. Pathol., 2001,20(4):359-367.
27	BRUSTMANN H. Expression of cellular apoptosis susceptibility protein in serous ovarian carcinoma: a clinicopathologic and immunohistochemical study [J]. Gynecol. Oncol., 2004,92(1):268-276.
28	TAI C J, HSU C H, SHEN S C, et al.. Cellular apoptosis susceptibility (CSE1L/CAS) protein in cancer metastasis and chemotherapeutic drug-induced apoptosis [J/OL].J. Exp. Clin. Cancer Res.,2010,29(1):110 [2024-07-19]. .
29	LIU W J, ZHOU Z Q, LI Y, et al.. CSE1L silencing impairs tumor progression via MET/STAT3/PD-L1 signaling in lung cancer [J]. Am. J. Cancer. Res., 2021,11(9):4380-4393.
30	LORENZATO A, BIOLATTI M, DELOGU G, et al.. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells [J]. Exp. Cell Res., 2013, 319(17): 2627-2636.
31	TAI C J, SU T C, JIANG M C, et al.. Correlations between cytoplasmic CSE1L in neoplastic colorectal glands and depth of tumor penetration and cancer stage [J]. J. Transl Med., 2023,21(1):315 [2024-07-19]. .
32	LIU X, LI X, GUAN K. Effect of CSE1L silencing on cytarabine sensitivity of acute myeloid leukemia cells [J]. Chin. J. Pathophysiol., 2023, 39(2): 269-275.

[1]	Qiangzhou WANG, Shiyu PAN, Mengya FANG, Wei LI, Jiaxing WANG, Yinyin LIU, Shihao CHEN. Construction of Chicken Embryo Fibroblast Cell Line with TET2 Gene Knockout Based on CRISPR-Cas9 Technology [J]. Journal of Agricultural Science and Technology, 2023, 25(11): 227-233.
[2]	Chundi XIE, Xiaorong GUO, Xudong ZHANG, Rong ZHOU, Kui LI. Establishment of IGF2R Knockout PK-15 Cells by CRISPR/Cas9 System [J]. Journal of Agricultural Science and Technology, 2022, 24(10): 200-207.
[3]	Zhong ZHUANG, Long ZHAO, Hao BAI, Yulin BI, Yingquan HUANG, Guohong CHEN, Guobin CHANG. Development of CRISPR/Cas9 and Its Application in Poultry [J]. Journal of Agricultural Science and Technology, 2022, 24(1): 14-23.
[4]	LIAO Jiaming, §, LI Chunmei§, ZHANG Shihu, LI Buye, OUYANG Kunxi, CHEN Xiaoyang. Development of CRISPR/Cas9 System and Its Aapplication in Plants [J]. Journal of Agricultural Science and Technology, 2021, 23(12): 20-28.
[5]	XIN Hongjia, LI Pengcheng, TENG Shouzhen, LI Shengyan, WANG Hai, LANG Zhihong*. Construction and Functional Characterization of Mutants of Arabidopsis SWEET1/2/3 Genes [J]. Journal of Agricultural Science and Technology, 2020, 22(2): 39-49.
[6]	LIU Dujuan, HUANG Huoqing, SU Xiaoyun. Construction of a Trichoderma reesei Strain with Low Cellulase Background and Its Application [J]. Journal of Agricultural Science and Technology, 2020, 22(12): 50-57.
[7]	HAN Song, DUAN Xingpeng, ZHANG Zhidong, ZUO Kaijing*. Functional Verification and Pathogenesis Analysis of VdTri15 Gene from Verticillium dahliae on Cotton [J]. Journal of Agricultural Science and Technology, 2018, 20(12): 26-35.