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Biologia

使用 CRISPR/Cas9 系统生成着丝粒相关蛋白- E CENP-E-/- 敲除细胞系

Published: June 23, 2023
doi:
10.3791/65476
, , , , , , , ,
1Department of Cell Biology and Genetics, The School of Basic Medical Sciences,Fujian Medical University, 2Key Laboratory of Stem Cell Engineering and Regenerative Medicine,Fujian Province University, 3College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics,Fujian Medical University, 4Medical Research Center,Fujian Maternity and Child Health Hospital

Summary

本文报道了使用 CRISPR/Cas9 系统和三种基于表型的筛选策略构建着丝粒相关蛋白-E （CENP-E） 敲除细胞的过程。我们利用 CENP-E基因敲除细胞系建立了一种新的方法来验证CENP-E抑制剂的特异性和毒性，这对药物开发和生物学研究很有用。

Abstract

CRISPR（成簇的规则间隔短回文重复序列）/Cas9 系统已成为在各种生物体中进行精确和高效基因编辑的有力工具。着丝粒相关蛋白-E （CENP-E） 是着丝粒-微管捕获、染色体比对和纺锤体组装检查点所需的正端定向驱动蛋白。尽管 CENP-E 蛋白的细胞功能已得到充分研究，但使用传统方案研究 CENP-E 蛋白的直接功能一直很困难，因为 CENP-E 消融通常会导致纺锤体组装检查点激活、细胞周期停滞和细胞死亡。在这项研究中，我们完全敲除了人类HeLa细胞中的 CENP-E 基因，并利用CRISPR/Cas9系统成功生成了 CENP-E-/- HeLa细胞。

建立了细胞集落筛选、染色体比对表型、CENP-E蛋白荧光强度等3种基于表型的优化筛选策略，有效提高了 CENP-E 敲除细胞的筛选效率和实验成功率。重要的是， CENP-E 缺失会导致染色体错位、BUB1 有丝分裂检查点丝氨酸/苏氨酸激酶 B （BubR1） 蛋白的异常位置和有丝分裂缺陷。此外，我们还利用 CENP-E 敲除HeLa细胞模型开发了CENP-E特异性抑制剂的鉴定方法。

在这项研究中，已经建立了一种验证 CENP-E 抑制剂特异性和毒性的有用方法。此外，本文还介绍了使用CRISPR/Cas9系统 进行CENP-E 基因编辑的方案，这可能是研究CENP-E在细胞分裂中机制的有力工具。此外， CENP-E基因敲除细胞系将有助于CENP-E抑制剂的发现和验证，对抗肿瘤药物开发、细胞生物学中细胞分裂机制的研究以及临床应用具有重要意义。

Introduction

工程基因组编辑介导各种细胞和生物体中基因的靶向修饰。在真核生物中，可以通过应用序列特异性核酸酶来引入位点特异性诱变，这些核酸酶刺激靶 DNA1 的同源重组。近年来，几种基因组编辑技术，包括锌指核酸酶 （ZFNs）2,3、转录激活因子样效应核酸酶 （TALENs）4,5 和归巢巨核酸酶 6,7，已被设计用于在特定位点切割基因组，但这些方法需要复杂的蛋白质工程和冗余的实验程序。研究表明，II 型原核生物簇状规则间隔短回文重复序列 （CRISPR）/Cas 系统是一种高效的基因编辑技术，可特异性介导多种细胞和物种中的 RNA 引导的位点特异性 DNA 切割 8,9,10,11。CRISPR/Cas9 基因敲除技术彻底改变了基础生物学、生物技术和医学领域12。

细菌和大多数古细菌已经进化出一种基于RNA的适应性免疫系统，该系统使用CRISPR和Cas蛋白来识别和破坏病毒和质粒13。化脓性链球菌Cas9 （SpCas9） 核酸内切酶含有 RuvC 样 Holliday 连接解析酶 （RuvC） 和 His-Asn-His （HNH） 结构域，可通过提供含有 CRISPR RNA （crRNA） 和反式激活 crRNA （tracrRNA） 的合成单向导 RNA （sgRNA） 有效介导序列特异性双链断裂 （DSB）14,15,16.DSB 可以通过插入缺失形成的非同源末端连接 （NHEJ） 或同源定向修复 （HDR） 通路进行修复，该通路在哺乳动物细胞中引入多种突变，包括插入、缺失或无疤痕的单核苷酸替换 1,8。容易出错的 NHEJ 和高保真 HDR 通路都可用于通过插入或缺失介导基因敲除，这可能导致移码突变和过早终止密码子10。

Kinesin-7 CENP-E 是细胞分裂过程中着丝粒-微管附着和染色体排列所必需的 17,18,19。CENP-E 的抗体显微注射20、21、siRNA 耗竭22、23、化学抑制24、25、26 和基因缺失27、28、29 导致染色体错位、纺锤体组装检查点和有丝分裂缺陷的激活，从而导致非整倍体和染色体不稳定19,30.在小鼠中，CENP-E缺失导致发育异常和胚胎致死率在发育的早期阶段27,29,31。CENP-E的基因缺失通常会导致染色体错位和细胞死亡26,27,29，这是研究CENP-E蛋白的功能和机制的障碍。

最近的一项研究使用生长素诱导的 CRISPR/Cas9 基因编辑方法32 建立了条件性 CENP-E 敲除细胞系，该方法可以在相对较短的时间内快速降解 CENP-E 蛋白33。然而，迄今为止，尚未建立稳定的CENP-E敲除细胞系，这是CENP-E生物学中尚未解决的技术挑战。考虑到遗传稳健性34、遗传补偿反应35、36、37和复杂的细胞内环境，由于CENP-E完全缺失的直接后果可能是复杂和不可预测的，因此建立CENP-E敲除细胞系对于研究染色体排列、纺锤体组装检查点和下游信号通路的机制非常重要。

CENP-E抑制剂的发现和应用对癌症治疗具有重要意义。迄今为止，已经发现并合成了七种类型的CENP-E抑制剂，包括GSK923295及其衍生物24,25、PF-2771 38,39、咪唑并[1,2-a]吡啶支架衍生物40,41、化合物A42,43、syntelin44,45、UA6278446和苯并[d]吡咯并[2,1-b]噻唑衍生物47.在这些抑制剂中，GSK923295是一种变构且有效的 CENP-E 抑制剂，它与 CENP-E 的运动结构域结合并抑制 CENP-E 微管刺激的 ATP 酶活性，Ki 为 3.2 ± 0.2 nM24,25。然而，与GSK923295对培养的癌细胞的抑制作用相比，GSK923295在临床癌症患者中的治疗效果并不理想48,49，这也引起了人们对GSK923295对CENP-E特异性的担忧。此外，其他CENP-E抑制剂对CENP-E蛋白的特异性和副作用是癌症研究的关键问题。

在这项研究中，我们使用 CRISPR/Cas9 系统完全敲除了 HeLa 细胞中的 CENP-E 基因。建立了细胞集落筛选、染色体比对表型、CENP-E蛋白荧光强度等3种基于表型的优化筛选策略，提高了CENP-E基因编辑的筛选效率和成功率。此外， CENP-E 敲除细胞系可用于测试CENP-E候选化合物的特异性。

Protocol

1. CRISPR/Cas9基因敲除载体的构建 选择人类 CENP-E 基因上的靶基因组DNA序列（GenBank登录号。NM_001286734.2） 并使用在线 CRISPR 设计工具 （http://crispor.tefor.net/） 设计 sgRNA。 输入单个基因组序列，选择“Homo sapiens-human-UCSC Dec 2013 （hg38 analysis set） + single nucleotide polymorphisms （SNPs）： dbSNP148”的基因组，选择原间隔区相邻基序“20 bp-NGG-spCas9”。根据特异性评分<sup c…

Representative Results

使用CRISPR/Cas9系统成功生成了CENP-E-/-HeLa细胞（图1）。该方法的时间表和关键实验步骤如图1所示。首先，我们设计合成了CENP-E特异性sgRNA，将sgRNA退火并连接到pX458质粒中，将质粒转染到HeLa细胞中，并培养48 h。将转染的细胞解离并使用连续稀释液接种在96孔板中（图1A）。使用三种基于表型的策略选择和筛选单…

Discussion

驱动蛋白-7 CENP-E 是细胞分裂过程中染色体排列和纺锤体组装检查点的关键调节因子 17,19,20。CENP-E 的基因缺失通常会导致纺锤体组装检查点的激活、细胞周期停滞和细胞死亡 27,29,51,52。因此，构建稳定的CENP-E敲除细胞系是CENP…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢福建医科大学细胞骨架实验室的所有成员进行了有益的讨论。我们感谢福建医科大学公共技术服务中心的Jun-Jin Lin、Zhi-Hong Huang、Ling Lin、Li-Li Pang、Lin-Ying 周、习 Lin和Min-Xia Wu的技术援助。我们感谢福建医科大学基础医学实验教学中心的郑思义、林颖和齐克的支持。本研究得到了以下资助：国家自然科学基金（批准号82001608和82101678）、中国福建省自然科学基金（批准号：2019J05071）、中国福建省科技创新联合基金（批准号：2021Y9160）和福建医科大学高层次人才科研启动资助项目（批准号XRCZX2017025）。

Materials

0.25% Trypsin-EDTA Gibco 25200056
1.5 mL centrifuge tube Axygen MCT-150-C
24-well plate Corning 3524
4S Gelred, 10,000x in water Sangon Biotech (Shanghai) A616697
50 mL centrifuge tube Corning 430828
6 cm Petri dish Corning 430166
95% ethanol Sinopharm Chemical Reagent 10009164
96-well plate Corning 3599
Acetic acid Sinopharm Chemical Reagent 10000218 Dissolve in H2O to prepare a 10% working solution.
Agarose Sangon Biotech (Shanghai) A620014
Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L) Beyotime A0428 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L) Beyotime A0423 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L) Beyotime A0460 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Anhydrous ethanol Sinopharm Chemical Reagent 100092690
Anti-BubR1 rabbit monoclonal antibody Abcam ab254326 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution 
Anti-CENP-B mouse monoclonal antibody Santa Cruz Biotechnology sc-376392 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution.
Anti-CENP-E rabbit monoclonal antibody Abcam ab133583 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Anti-fade mounting medium Beyotime P0131 Slowing down the quenching of fluorescent signals.
Anti-α-tubulin mouse monoclonal antibody Abcam ab7291 For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Biotek Epoch Microplate Spectrophotometer Biotek Instruments Biotek Epoch
Bovine Serum Albumin (BSA) Sinopharm Chemical Reagent 69003435
BpiI (BbsI) Thermo Fisher Scientific ER1011
CellTiter 96 aqueous one solution cell proliferation assay Promega G3580
Centrifuge Eppendorf 5424BK745380
Colchicine Sinopharm Chemical Reagent 61001563
Confocal scanning microscope Leica Leica TCS SP8
Coverslip CITOTEST 80344-1220
DAPI Beyotime C1006
DH5α competent cells Sangon Biotech (Shanghai) B528413
DL2000 DNA marker TaKaRa 3427A
Dulbecco's Modified Eagle Medium (DMEM) Gibco C11995500BT
Endo-free plasmid mini kit Equation 2 Omega D6950
Ezup Column Animal Genomic DNA Purification Kit Sangon Biotech (Shanghai) B518251
Fetal bovine serum Zhejiang Tianhang Biotechnology 11011-8611
Gentian violet Sinopharm Chemical Reagent 71019944 Dissolve in PBS to prepare 0.1% gentian violet/PBS.
Giemsa staining solution Sinopharm Chemical Reagent 71020260
GraphPad Prism version 8.0 software GraphPad www.graphpad.com Statistical analysis.
GSK923295 MedChemExpress HY-10299
HeLa cell line ATCC CCL-2
Humidified incubator Heal Force HF90/HF240
Image J software National Institutes of Health https://imagej.nih.gov/ij/ Image processing and analysis.
Inverted microscope Nanjing Jiangnan Novel Optics XD-202
LB agar powder Sangon Biotech (Shanghai) A507003
Lipo6000 transfection reagent Beyotime C0526
Nikon Ti-S2 microscope Nikon Ti-S2
Opti-MEM reduced serum medium Gibco 31985070
Paraformaldehyde Sinopharm Chemical Reagent 80096618 Dissolve in PBS to prepare 4% paraformaldehyde/PBS.
Penicillin-streptomycin solution HyClone SV30010
SanPrep column DNA gel extraction kit Sangon Biotech (Shanghai) B518131
SanPrep column plasmid mini-preps kit Sangon Biotech (Shanghai) B518191
T4 DNA ligase TaKaRa 2011A
T4 polynucleotide kinase TaKaRa 2021A
TaKaRa Ex Taq TaKaRa RR001A
Triton X-100 Sinopharm Chemical Reagent 30188928 Dissolve in PBS to prepare 0.25% Triton X-100/PBS.
Tween 20 Sinopharm Chemical Reagent 30189328 Dissolve in PBS to prepare 0.1% Tween 20/PBS.
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Keywords: CENP-ECRISPR/Cas9KnockoutHeLa CellsChromosome AlignmentMitotic DefectsCell Cycle ArrestCell DeathKinetochore-microtubule CaptureSpindle Assembly Checkpoint
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Xu, M., Chen, J., Xu, Y., Zhang, J., Zhou, Y., He, J., Wu, S., Wei, Y., She, Z. Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System. J. Vis. Exp. (196), e65476, doi:10.3791/65476 (2023).
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