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）。

1. CRISPR/Cas9基因敲除载体的构建
<ol><li>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。</li>
	<li>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50分、预测效率10分和最小脱靶率，选择sgRNA-1、5'-CGGCCGCACTCGCACGCAGA-3'和sgRNA-2 5'-TTCTTTAGAGACGCGGGCTC-3'两个引导序列。</li>
	<li>订购并合成单链 DNA 寡核苷酸 （ssODN）。将ddH2O中的ssODN稀释至终浓度为100μM。为了磷酸化和退火sgRNA寡核苷酸，在聚合酶链反应（PCR）管中加入1μL正向寡核苷酸，1μL反向寡核苷酸，0.5μLT4多核苷酸激酶，1μLT4多核苷酸激酶缓冲液和6.5μL无RNase水，并在37°C孵育管30分钟，95°C孵育5分钟;然后，在5°C min-1下升温至25°C。</li>
	<li>使用 Bbs<img alt="Equation 1" src="/files/ftp_upload/65476/65476eq01.jpg" style="margin: auto;"> 限制性内切酶消化 pX458 质粒。加入 1 μg pX458 质粒、2 μL 10x 缓冲液 G、1 μL Bbs<img alt="Equation 1" src="/files/ftp_upload/65476/65476eq01.jpg" style="margin: auto;">，并在 1.5 mL 离心管中加入无 RNase ddH2O 至 20 μL。将管在37°C孵育2小时。然后，根据制造商的方案，使用柱DNA凝胶提取试剂盒纯化线性质粒。</li>
	<li>将 sgRNA 寡核苷酸连接到 pX458 质粒中（pSpCas9（BB）-2A-绿色荧光蛋白 （GFP） 质粒，Addgene ID. 48138）。加入 1 μL 退火寡核苷酸、25 ng 线性质粒、1 μL 10x T4 DNA 连接缓冲液、0.5 μL T4 DNA 连接酶 （350 U/μL），并在 1.5 mL 离心管中用 ddH2O 配制多达 10 μL。将连接溶液在16°C孵育2小时。</li>
	<li>将10μL构建的质粒与感受态DH5α细胞在冰上孵育30分钟，在42°C热休克45秒，并立即将它们放在冰上5分钟。加入 500 μL Luria-Bertani （LB） 培养基，并将细胞接种在含有氨苄青霉素 （100 μg/mL） 的 LB 平板上。将其在37°C孵育16小时并筛选抗性克隆。</li>
	<li>用灭菌的移液器吸头选择 5-10 个单菌落，并将它们转移到含有氨苄青霉素 （100 μg/mL） 的 1 mL LB 培养基中。将培养物在37°C孵育，并以180rpm振荡12-16小时。</li>
	<li>根据制造商的方案，使用质粒提取试剂盒分离质粒DNA。使用 U6-Fwd 引物 5'-GAGGGCCTTCCCATGATTCC-3' 通过 U6 启动子位点的 Sanger 测序验证每个菌落的质粒 DNA。</li>
	<li>根据制造商的方案，使用无内切质粒DNA试剂盒提取和纯化质粒。</li>
</ol>2. CENP-E 敲除HeLa细胞的转染、分离和筛选
<ol><li>在37°C和5%CO2的加湿培养箱中，在补充有10%胎牛血清和1%青霉素/链霉素的Dulbecco改良Eagle培养基（DMEM）中培养HeLa细胞。</li>
	<li>使用0.25%胰蛋白酶/乙二胺四乙酸（EDTA）在37°C孵育HeLa细胞2-3分钟，通过轻轻移液解离细胞，然后以1：4的稀释比例将细胞接种在新板中用于细胞传代。</li>
	<li>将细胞接种在 12 孔板中，并在转染前培养至 60-70% 汇合度。根据制造商的方案，使用参考试剂将pX458-sgRNA质粒转染到HeLa细胞中（参见 材料表）。<ol><li>在试管 A 中轻轻混合 1 μg 经验证的 pX458-sgRNA 质粒和 50 μL 还原血清培养基。然后，在B管中轻轻混合2μL转染试剂和50μL还原血清培养基，在室温下分别孵育A和B管5分钟。</li>
		<li>轻轻混合试管 A 和试管 B，并在室温下孵育 5 分钟。将混合物加入12孔板中，孵育细胞6小时，并用新鲜的DMEM培养基代替培养基。</li>
	</ol></li>
	<li>在24小时或48小时后使用荧光显微镜检查HeLa细胞的转染效率。转染48小时后，在37°C下使用0.25%胰蛋白酶/ EDTA完全解离转染的HeLa细胞3-5分钟，使用Neubauer室或自动细胞计数器计数细胞数，并根据连续稀释方法10将细胞铺板到每个转染群体的三个单独的96孔板中。</li>
	<li>将细胞放回湿培养箱中，然后再培养 1-2 周。每 3 天更换一次培养基。对于基于表型的 CENP-E 敲除筛选和验证，在 24 孔或 12 孔板中解离并扩增细胞 5-7 天。</li>
	<li>使用配备10倍和20倍物镜的倒置显微镜筛选细胞集落直径较小的突变细胞。 注意： CENP-E 突变通常会导致细胞集落中分裂细胞数量的显着增加，这可能是筛选 CENP-E 突变细胞的关键指标之一。</li>
	<li>根据制造商的方案，使用柱动物基因组DNA提取试剂盒收获用于DNA提取的细胞。按如下方式设置PCR反应：0.25μL Taq聚合酶，5μL 10x缓冲液（Mg2+ plus），4μL dNTP混合物，500ng DNA模板，1μL正向寡核苷酸，1μL反向寡核苷酸，并将ddH2O调节至50μL。 使用以下PCR程序设置进行基因扩增： 98°C，10秒;98 °C、10 s、55 °C、30 s、72 °C、60 s 共 33 个循环;72°C，10分钟，然后在4°C下保持。 注：用于克隆靶位点的特异性引物如下： CENP-E 靶标F1,5'-GAGGGTCCTGGCCATTTTCCTG-3'; CENP-E 靶标R1,5'-AGATCTCCGATCCTCCCCTGTC-3'; CENP-E 靶标F2,5'-TGGTAACTGCATTTTGGTGTTCTAC-3'; CENP-E 靶标R2,5'-CCTGTTGCAACGTGAGGGAAG-3'。</li>
	<li>根据制造商的方案将靶DNA连接到pMD18-T载体中。将连接的质粒转染到感受态DH5α细胞中并培养用于选择克隆。</li>
	<li>执行 Sanger 测序以确定 CENP-E 敲除的类型。根据制造商的方案，使用质粒提取试剂盒分离质粒DNA。使用 M13 正向和反向引物对每个菌落的质粒 DNA 进行 Sanger 测序。M13F引物，5'-TGTAAAACGACGGCCAGT-3';M13R引物，5'-CAGGAAACAGCTATGACC-3'。</li>
	<li>将野生型和 CENP-E 突变HeLa细胞分别接种在24孔板中的12mm玻璃盖玻片上。除去完整的DMEM培养基，并在室温下将细胞固定在4%多聚甲醛/磷酸盐缓冲盐水（PBS）固定溶液中10分钟。</li>
	<li>在室温下用4'，6-二脒基-2-苯基吲哚（DAPI）溶液染色细胞核5分钟。使用配备 Plan Fluor 40x/数值孔径 （NA） 0.75 物镜的荧光显微镜，根据染色体比对的表型筛选和验证 CENP-E 突变细胞。</li>
	<li>使用CENP-E蛋白的免疫荧光染色和分析（参见第3节）筛选和验证CENP-E敲除细胞。</li>
</ol>3. 免疫荧光染色和高分辨率共聚焦显微镜
<ol><li>用4%多聚甲醛/ PBS固定溶液在室温下固定细胞10分钟，并在1x PBS中浸入2×5分钟。 注意：确保细胞处于健康状态，并轻轻收集和固定细胞，以避免中期细胞脱离。</li>
	<li>在室温下用0.25%Triton X-100 / PBS透化细胞10分钟，并将其浸入1x PBS中2×5分钟。</li>
	<li>在室温下用1%BSA / PBST（PBS中的0.1%吐温20）封闭细胞1小时。用1%BSA / PBST稀释一抗，并将样品在4°C孵育12小时。</li>
	<li>弃去一抗溶液，用1x PBS冲洗细胞3×5分钟，并在室温下用稀释的二抗孵育细胞2小时。</li>
	<li>弃去二抗，在1x PBS中冲洗细胞3×5分钟。</li>
	<li>在室温下用DAPI染色细胞核5分钟。使用安装介质安装载玻片。用指甲油密封载玻片。</li>
	<li>使用配备63x / NA 1.40物镜的扫描共聚焦显微镜观察和记录荧光图像。</li>
</ol>4. 染色体制备和核型分析
<ol><li>在补充有 10% 胎牛血清和 1% 青霉素/链霉素的完全 DMEM 培养基中培养野生型和 CENP-E-/- HeLa 细胞 24 小时，然后将野生型和 CENP-E-/- HeLa 细胞与 300 nM 秋水仙碱孵育 5 小时。</li>
	<li>将细胞与0.25%胰蛋白酶/ EDTA在37°C下孵育3分钟，并将细胞收集到1.5mL离心管中。</li>
	<li>在室温下以1,000× g 离心细胞5分钟，弃去上清液，加入1.2mL0.075mol/L KCl溶液，并在37°C孵育20分钟。</li>
	<li>在室温下以 1,000 × g 离心细胞 5 分钟，弃去上清液，加入 0.2 mL 固定溶液（甲醇：冰醋酸 = 3：1）用于预固定，并轻轻混合 1 分钟。</li>
	<li>在室温下以1,000× g 离心细胞5分钟，收集沉淀，在室温下加入1.5mL固定溶液10分钟，然后在室温下以1,000× g 离心5分钟。</li>
	<li>弃去上清液，加入 0.6 mL 固定溶液，并制备细胞悬浮液。在载冰上从35-40厘米的高度加入3-5滴细胞悬浮液。 注意：将细胞悬液释放到载玻片上的高度保持在35-40厘米;否则，染色体可能过于分散或彼此之间没有分离。</li>
	<li>立即用酒精灯干燥载玻片，用10%Giemsa染色溶液染色样品7分钟，用流水冲洗载玻片2分钟，并使用配备Plan Fluor 40x / NA 0.75物镜的光学显微镜观察样品。</li>
</ol>5. 细胞集落形成测定
<ol><li>在6孔板中以1,000-2,000个细胞/孔的细胞密度制备细胞悬液。轻轻摇晃板以均匀分布细胞。</li>
	<li>将6孔板放入CO2 培养箱中，并在37°C下用5%CO2孵育2-3周。每 5 天更换一次培养基。使用配备10倍和20倍物镜的倒置显微镜记录图像。当菌落可见时收获细胞（克隆内有数百个细胞）。</li>
	<li>为了研究GSK923295的影响，在6孔板中培养细胞24小时，并加入2mL终浓度为10、25、50、100和200nM的GSK923295。 注意：不要在克隆形成的早期阶段移动培养皿，以避免细胞脱落，这可能会形成新的克隆并影响转染细胞的筛选。</li>
	<li>弃去培养基，并在室温下用1%PFA固定细胞10分钟。在室温下用0.1%结晶紫染色溶液染色菌落15分钟。用 1x PBS 冲洗样品三次。使用 ImageJ 软件记录细胞菌落的图像并量化每个菌落的直径。选择 直线 选择工具，单击 分析，选择 测量，然后记录长度。</li>
	<li>用 1 mL 10% 乙酸溶液冲洗菌落 5 分钟。在 96 孔板中转移 200 μL 上清液溶液，并使用微孔板分光光度计在A 600 nm 处测量吸光度值。</li>
</ol>6. 细胞活力测定
<ol><li>将细胞接种在24孔板中，并将细胞培养48小时至80-90%密度。</li>
	<li>将细胞与100μL0.25%胰蛋白酶/ EDTA在37°C孵育3分钟。</li>
	<li>用移液管枪将 400 μL PBS 与细胞混合。将 100 μL 细胞悬液转移到 96 孔板中。</li>
	<li>在每个孔中加入 20 μL MTS 溶液，终浓度为 317 μg/mL，并根据制造商的方案轻轻混合。将样品在37°C下在CO2 培养箱中孵育1-3小时。</li>
	<li>使用微孔板分光光度计记录A490nm 吸光度峰处每个孔的吸光度值。使用 A630 nm 的参考波长减去细胞碎片和非特异性吸光度（细胞活力 =A 490nm- A630 nm）贡献的背景。</li>
</ol>

使用 CRISPR/Cas9 系统进行着丝粒相关 Protein-E 基因编辑

作者没有需要披露的利益冲突。

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>4S Gelred, 10,000x in water</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A616697</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10009164</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10000218</td>
			<td>Dissolve in H2O to prepare a 10% working solution.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A620014</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0423</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0460</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>100092690</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BubR1 rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab254326</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution&#160;</td>
		</tr>
		<tr>
			<td>Anti-CENP-B mouse monoclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376392</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution.</td>
		</tr>
		<tr>
			<td>Anti-CENP-E rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab133583</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Anti-fade mounting medium</td>
			<td>Beyotime</td>
			<td>P0131</td>
			<td>Slowing down the quenching of fluorescent signals.</td>
		</tr>
		<tr>
			<td>Anti-&#945;-tubulin mouse monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Biotek Epoch Microplate Spectrophotometer</td>
			<td>Biotek Instruments</td>
			<td>Biotek Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>69003435</td>
			<td></td>
		</tr>
		<tr>
			<td>BpiI (BbsI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER1011</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter 96 aqueous one solution cell proliferation assay</td>
			<td>Promega</td>
			<td>G3580</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424BK745380</td>
			<td></td>
		</tr>
		<tr>
			<td>Colchicine</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>61001563</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal scanning microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>CITOTEST</td>
			<td>80344-1220</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B528413</td>
			<td></td>
		</tr>
		<tr>
			<td>DL2000 DNA marker</td>
			<td>TaKaRa</td>
			<td>3427A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo-free plasmid mini kit <img alt="Equation 2" src="/files/ftp_upload/65476/65476eq02.jpg" style="margin: auto;" /></td>
			<td>Omega</td>
			<td>D6950</td>
			<td></td>
		</tr>
		<tr>
			<td>Ezup Column Animal Genomic DNA Purification Kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518251</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Zhejiang Tianhang Biotechnology</td>
			<td>11011-8611</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentian violet</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>71019944</td>
			<td>Dissolve in PBS to prepare 0.1% gentian violet/PBS.</td>
		</tr>
		<tr>
			<td>Giemsa staining solution</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>71020260</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 8.0 software</td>
			<td>GraphPad</td>
			<td>www.graphpad.com</td>
			<td>Statistical analysis.</td>
		</tr>
		<tr>
			<td>GSK923295</td>
			<td>MedChemExpress</td>
			<td>HY-10299</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cell line</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator</td>
			<td>Heal Force</td>
			<td>HF90/HF240</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Image processing and analysis.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nanjing Jiangnan Novel Optics</td>
			<td>XD-202</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar powder</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A507003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipo6000 transfection reagent</td>
			<td>Beyotime</td>
			<td>C0526</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti-S2 microscope</td>
			<td>Nikon</td>
			<td>Ti-S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM&#160;reduced&#160;serum&#160;medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80096618</td>
			<td>Dissolve in PBS to prepare 4% paraformaldehyde/PBS.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>SanPrep column DNA gel extraction kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518131</td>
			<td></td>
		</tr>
		<tr>
			<td>SanPrep&#160;column&#160;plasmid&#160;mini-preps&#160;kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518191</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>TaKaRa</td>
			<td>2011A</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>TaKaRa</td>
			<td>2021A</td>
			<td></td>
		</tr>
		<tr>
			<td>TaKaRa Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30188928</td>
			<td>Dissolve in PBS to prepare 0.25% Triton X-100/PBS.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30189328</td>
			<td>Dissolve in PBS to prepare 0.1% Tween 20/PBS.</td>
		</tr>
	</tbody>
</table>

generation of centromere-associated protein-e cenp-e-/- knockout cell lines using the crispr/cas9 system

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。

Engineered genome editing mediates the targeted modifications of genes in a variety of cells and organisms. In eukaryotes, site-specific mutagenesis can be introduced by the applications of sequence-specific nucleases that stimulate homologous recombination of target DNA1. In recent years, several genome editing technologies, including zinc finger nucleases (ZFNs)2,3, transcription activator-like effector nucleases (TALENs)4,5, and homing meganucleases6,7, have been engineered to cleave genomes at specific sites, but these approaches require complex protein engineering and redundant experimental procedures. Studies have shown that the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an efficient gene editing technology, which specifically mediates RNA-guided, site-specific DNA cleavage in a wide variety of cells and species8,9,10,11. The CRISPR/Cas9 gene knockout technology has revolutionized the fields of basic biology, biotechnology, and medicine12.
Bacteria and most archaea have evolved an RNA-based adaptive immune system that uses CRISPR and Cas proteins to identify and destroy viruses and plasmids13. Streptococcus pyogenes Cas9 (SpCas9) endonuclease contains the RuvC-like Holliday junction resolvase (RuvC) and His-Asn-His (HNH) domain, which can efficiently mediate sequence-specific, double-stranded breaks (DSBs) by providing a synthetic single-guide RNA (sgRNA) containing CRISPR RNAs (crRNA) and trans-activating crRNA (tracrRNA)14,15,16. DSBs can be repaired through the indel-forming non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, which introduces multiple mutations, including insertions, deletions, or scar-less single nucleotide substitutions, in mammalian cells1,8. Both the error-prone NHEJ and the high-fidelity HDR pathway can be used to mediate gene knockout through insertions or deletions, which can cause frameshift mutations and premature stop codons10.
Kinesin-7 CENP-E is required for kinetochore-microtubule attachment and chromosome alignment during cell division17,18,19. Antibody microinjection20,21, siRNA depletion22,23, chemical inhibition24,25,26, and genetic deletion27,28,29 of CENP-E leads to chromosome misalignment, the activation of spindle assembly checkpoint and mitotic defects, which results in aneuploidy and chromosomal instability19,30. In mice, CENP-E deletion results in abnormal development and embryonic lethality at the very early stages of development27,29,31. Genetic deletion of CENP-E usually leads to chromosome misalignment and cell death26,27,29, which is an obstacle in studying the functions and mechanisms of the CENP-E proteins.
A recent study has established a conditional CENP-E knockout cell line using an Auxin-inducible CRISPR/Cas9 gene-editing method32, which enables rapid degradation of CENP-E proteins in a relatively short time33. However, to date, stable CENP-E knockout cell lines have not been established, which is an unresolved technical challenge in CENP-E biology. Considering genetic robustness34, genetic compensation responses35,36,37, and complex intracellular environments, as the direct consequences of complete deletion of CENP-E may be complex and unpredictable, it is important to establish CENP-E knockout cell lines for the investigation of mechanisms of chromosome alignment, spindle assembly checkpoint, and downstream signaling pathways.
The discovery and applications of CENP-E inhibitors are important for cancer treatment. To date, seven types of CENP-E inhibitors have been found and synthesized, including GSK923295 and its derivatives24,25, PF-277138,39, imidazo[1,2-a]pyridine scaffold derivatives40,41, compound-A42,43, syntelin44,45, UA6278446, and benzo[d]pyrrolo[2,1-b]thiazole derivatives47. Among these inhibitors, GSK923295 is an allosteric and efficient CENP-E inhibitor that binds to the motor domain of CENP-E and inhibits CENP-E microtubule-stimulated ATPase activity with a Ki of 3.2 &#177; 0.2 nM24,25. However, compared with the inhibitory effects of GSK923295 on cultured cancer cells, the therapeutic effects of GSK923295 in clinical cancer patients are not ideal48,49, which also raised concerns about the specificity of GSK923295 for CENP-E. Moreover, the specificity and side effects of other CENP-E inhibitors on the CENP-E proteins are key issues in cancer research.
In this study, we have completely knocked out the CENP-E gene in HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies have been established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, to improve the screening efficiency and success rate of CENP-E gene editing. Furthermore, CENP-E knockout cell lines can be used to test the specificity of candidate compounds for CENP-E.

作者聚焦：建立 CENP-E 敲除 HeLa 细胞 – 一种研究驱动蛋白 7 CENP-E 生物学及其抑制剂的新方法

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

Genetic deletion of Kinesin-7 and CENP usually results in cell cycle arrest and cell death. The construction of stable CENP knockout cell lines is an important unresolved issue in CENP biology. In this study, we generated CENP knockout HeLa cells using a CRISPR/Cas9 system, and established three optimized screening strategies.Recently, several genome editing technologies, including zinc-finger nucleases, transcription activator-like effector nucleases, and how we make our nucleases, has been engineered to cleave genomes at a specific size. The CRISPR/Cas9 gene knockout technology has revolutionized basic biology, biotechnology, and medicine. CENP is essential for attaching kinetochores to microtubules and aligning chromosomes.Disrupting CENP, whether through antibody microinjection, sgRNA deletion, chemical inhibition, or genetic deletion, result in chromosome misalignment, mitotic defects, and often cell death, complicating the study of CENP protein mechanisms. In this study, three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment of phenotypes, and the fluorescent intensities of CENP proteins, which improved the screening efficiency and the experimental success rate. Importantly, CENP deletion results in chromosome misalignment, the abnormal location of BubR1 proteins, and mitotic defects.The interaction modes and the molecular mechanisms of CENP and its inhibitors are important research fields in cell biology and cancer research. In this study, the CENP knockout HeLa cell line has been established as a valuable tool for the validation of the specificity and the toxicity of CENP inhibitors.

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...

Watch this Scientific Journal Video about Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System at JoVE.com

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。

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

驱动蛋白-7 和 CENP 的基因缺失通常会导致细胞周期停滞和细胞死亡。稳定的CENP敲除细胞系的构建是CENP生物学中一个未解决的重要问题。在这项研究中，我们使用 CRISPR/Cas9 系统生成了 CENP 敲除 HeLa 细胞，并建立了三种优化的筛选策略。最近，几种基因组编辑技术，包括锌指核酸酶、转录激活因子样效应核酸酶，以及我们如何制造核酸酶，已被设计为切割特定大小的基因组。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。CENP 对于将动粒附着在微管上和排列染色体至关重要。无论是通过抗体显微注射、sgRNA 缺失、化学抑制还是基因缺失，破坏 CENP 都会导致染色体错位、有丝分裂缺陷，并经常导致细胞死亡，使 CENP 蛋白机制的研究复杂化。本研究建立了细胞集落筛选、表型染色体比对、CENP蛋白荧光强度等3种基于表型的优化筛选策略，提高了筛选效率和实验成功率。重要的是，CENP 缺失会导致染色体错位、BubR1 蛋白位置异常和有丝分裂缺陷。CENP及其抑制剂的相互作用模式和分子机制是细胞生物学和癌症研究的重要研究领域。在这项研究中，CENP 敲除 HeLa 细胞系已被确立为验证 CENP 抑制剂特异性和毒性的宝贵工具。

generation-centromere-associated-protein-e-cenp-e-knockout-cell-lines

Research

JoVE Journal

Biology

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

403 次观看.  Fujian Medical University. 驱动蛋白-7 和 CENP 的基因缺失通常会导致细胞周期停滞和细胞死亡。稳定的CENP敲除细胞系的构建是CENP生物学中一个未解决的重要问题。在这项研究中，我们使用 CRISPR/Cas9 系统生成了 CENP 敲除 HeLa 细胞，并建立了三种优化的筛选策略。最近，几种基因组编辑技术，包括锌指核酸酶、转录激活因子样效应核酸酶，以及我们如何制造核酸酶，已被设计为切割特定大小的基因?...

视频: 作者聚焦：建立 CENP-E 敲除 HeLa 细胞 – 一种研究驱动蛋白 7 CENP-E 生物学及其抑制剂的新方法

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

科研

生物学

Transfection, Isolation, and Screening of the CENP-E Knockout HeLa Cells

To begin, add 0.25%Trypsin-EDTA Solution to the HeLa cells and incubate at 37 degrees Celsius for two to three minutes. Gently dissociate the cells by pipetting and seed them in new plates at a ratio of one to four for cell passage. To transfect the plasmid into HeLa cells, mix one microgram of validated PX458 sgRNA plasmid, with 50 microliters of reduced serum medium in tube A.In tube B, gently mix two microliters of the transfection reagent and 50 microliters of reduced serum medium and incubate at room temperature for five minutes.Then, combine the contents from tubes A and B and incubate at room temperature for another five minutes. Add the mixture to the 12-well plate and incubate the cells at 37 degrees Celsius for six hours. At the end of the incubation, replace the medium with fresh DMEM medium.After 24 or 48 hours, assess the transfection efficiency by examining the HeLa cells under a fluorescence microscope. Dissociate the transfected HeLa cells fully using 0.25%trypsin EDTA and incubate at 37 degrees Celsius for three to five minutes. Next, count the cells using a Neubauer chamber or an automated cell counter.Plate the cells into three separate 96-well plates for each transfected population following serial dilution methods. Return the cells to a humidified incubator for one to two weeks. For phenotype-based screening and validation of CENP-E Knockout, dissociate and expand the cells in 24-well or 12-well plates for five to seven days.Screen the mutant cells with smaller colony diameters using an inverted microscope with 10 times and 20 times magnification objective lenses. Next, harvest the cells for DNA extraction by employing the Column Animal Genomic DNA Extraction Kit following the manufacturer's instructions and set up the polymerase chain reactions. Ligate the target DNA into the pMD18-T Vector as directed by the manufacturer's protocols.Transfect the ligated plasmid into competent DH5-Alpha cells, and proceed with the culture for clone selection. To identify the types of CENP-E Knockout, isolate the plasmid DNA with the help of a Plasmid Extraction Kit following the manufacturer's guidelines. Next, carry out Sanger sequencing for the plasmid DNA of each colony using M13 forward and reverse primers.Seed the wild type and CENP-E mutant HeLa cells on 12-millimeter glass cover slips in a 24-well plate. Remove the complete DMEM medium and fix the cells using a 4%paraformaldehyde and PBS solution at room temperature for 10 minutes. Next, stain the nuclei with DAPI at room temperature for five minutes.Observe and validate the CENP-E mutant cells based on chromosome alignment phenotype using a fluorescence microscope. The phenotypic screening of the colonies showed that the CENP-E Knockout cells showed increased chromosome misalignment compared to the control group.

CENP-E 敲除 HeLa 细胞的转染、分离和筛选

Immunofluorescence Staining and High-Resolution Confocal Microscopy of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

To begin, take a plate of wild type and CENP-E mutant HeLa cells growing on a cover slip. Fix the cells with 4%paraformaldehyde and PBS fixative solution at room temperature for 10 minutes. Then, immerse in PBS for two rounds of five minutes each.Next, permeabilize the cells with 0.25%Triton X-100 and PBS at room temperature for 10 minutes. Immerse them in PBS for two rounds of five minutes each. Proceed to block the cells with 1%BSA PBS with 0.1%Tween 20 at room temperature for one hour.Dilute the primary antibodies in 1%BSA and PBST and incubate the samples at four degrees Celsius for 12 hours. Discard the primary antibody solutions. Then, rinse the cells three times for five minutes each with PBS.Add diluted secondary antibodies to the cell and incubate at room temperature for two hours. Next, discard the secondary antibodies. Then rinse the cells with PBS three times for five minutes each.Stain the nuclei using DAPI at room temperature for five minutes. Mount the slides with the mounting medium and seal them with nail polish. Observe the fluorescence images using a scanning confocal microscope, fitted with a 63 times magnification, 1.40 numerical aperture objective, and record them for further analysis.Immunofluorescence staining of the control and CENP-E mutant groups showed that the fluorescence intensities of the CENP-E proteins were completely knocked out in the CENP-E mutant Clone 1 cells and significantly decreased in CENP-E mutant Clone 3 cells. The ratios of metaphase cells with chromosome misalignment increased in Clones 1 and 3 compared to the control cells. Meanwhile, the ratios of metaphase cells increased significantly in Clone 1 cells and Clone 3 cells.In addition, the ratio of interphase cells slightly increased in Clone 1 and 3 groups after CENP-E deletion, compared with the control group. The rates of prophase, anaphase, and telophase cells were slightly decreased after CENP-E deletion, whereas the rates of metaphase cells increased after CENP-E deletion.

CRISPR/Cas9介导的 CENP-E 敲除HeLa细胞的免疫荧光染色和高分辨率共聚焦显微镜

Chromosome Preparation and Karyotype Analysis of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

To begin, take the wild type and CENP-E mutant HeLa cell cultures and add 300 nanomolar colchicine before incubating them for five hours. Next, incubate the cells with 0.25%trypsin-EDTA at 37 degrees Celsius for three minutes, and collect the cells into 1.5 milliliter centrifuge tubes. Next, centrifuge the cells at 1000 G for five minutes at room temperature, discard the supernatants and add 1.2 milliliters of 0.075 molar potassium chloride solution.Incubate the cells at 37 degrees Celsius for 20 minutes. At the end of the incubation, centrifuge the cells, discard the supernatant, and add 0.2 milliliters of fixative solution for prefix. Mix gently for one minute, then collect the cell pellets as demonstrated earlier, and add 1.5 milliliters of fixative solution at room temperature for 10 minutes.After centrifuging the cells and discarding the supernatant, add 0.6 milliliters of the fixative solutions to create a cell suspension. Carefully drop three to five droplets of the cell suspensions from 35 to 40 centimeters onto the ice slides. Immediately dry the slides using an alcohol lamp and stain them with 10%Giemsa's sustaining solution for seven minutes.Rinse the slides with running water for two minutes, then examine the samples using a light microscope equipped with a Plan Fluor 40 times magnification 0.75 numerical aperture objective. The karyotype analysis revealed decreased chromosome numbers in the CENP-E mutant HeLa cells compared with the wild type cells.