CN105567689A - CRISPR/Cas9靶向敲除人TCAB1基因及其特异性gRNA - Google Patents
CRISPR/Cas9靶向敲除人TCAB1基因及其特异性gRNA Download PDFInfo
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Abstract
本发明属于分子生物学与生物医学技术领域,具体的说,本发明涉及基于CRISPR/Cas9系统的gRNA序列在引起肿瘤细胞凋亡中的应用。本发明根据CRISPR/Cas9的设计原则,在人基因组上设计了两个靶位点,合成相应的oligos,并且将其构建在px458载体上。在人肝癌细胞株(HepG2)中利用针对这两个靶点设计构建gRNA指导的CRISPR/Cas9系统,可以有效的敲除人TCAB1基因,这一系统易于操作,人TCAB1基因敲除效率高。本发明涉及的gRNA指导CRISPR/Cas9系统有望在治疗肿瘤的新型药物中得到应用。
Description
技术领域
本发明属于分子生物学与生物医学技术领域,具体涉及基于CRISPR/Cas9系统的gRNA序列在促进肿瘤细胞凋亡中的应用。
背景技术
CRISPR/Cas系统是从细菌和古生菌对抗外来病毒或质粒的适应性免疫系统发展而来,包括三种不同的类型,其中TypeII型的CRISPR/Cas系统的DNA内切酶Cas9只有一个亚基,结构最为简单,所以应用也最广泛。除了Cas9蛋白外,该系统还包括两条短的CRISPRRNAs(crRNAs)和trans-activatingcrRNAs(tracrRNA)。成熟的crRNA-tracrRNA复合体可以通过碱基互补配对指导Cas9蛋白到靶序列上,并在PAM(protospaceradjacentmotif)附近特异性剪切DNA双链,形成DSB(doublestrandbreak)。DSB可以通过两种途径被修复,一种是非同源重组的末端接合(Non-HomologousEndJoiningNHEJ)DNA修复方式,另一种是同源重组修复(HomologyDirectedRepairHDR)方式。NHEJ修复方式可能产生碱基的插入或缺失,从而产生移码突变,或者也可能突变成终止密码子,这些突变形式都可以改变目的基因的开放阅读框;HDR方式需要一段与被剪切片段同源的模板片段来修复DSB,这种修复方式可以将被用来作为模板的同源片段的序列复制到目的基因中,所以可以利用这种修复方式将特定的基因片段引入到目的基因中。
端粒是染色体末端的一段六核苷酸DNA重复序列,被认为是一段沉默基因,它在染色体末端形成类似保护帽结构,避免染色体从末端双链DNA被核酸酶水解,维持遗传物质的稳定性和干细胞及肿瘤细胞的无限增值潜能。而端粒DNA由端粒酶催化合成,合成机制涉及一系列复杂过程,包括酶的生物合成组装、全酶复合物核内的转移定位和活性调控等。端粒酶是一种具有端粒重复序列的核糖核蛋白复合物,它能够识别端粒特定重复序列,并以自身RNA序列为模板,以逆转录的形式合成新的端粒序列以补偿有丝分裂过程中丢失的端粒DNA。
端粒和端粒酶作为维持染色体的稳定的主要结构和细胞寿命的“生物钟”,对于恶性肿瘤的细胞永生化起到重要作用。当细胞发生癌变时,端粒DNA随分裂活动发生渐进性缩短的趋势会启动端粒酶,在端粒酶的作用下,正常的人体细胞可转化成具有无限增值能力的永生化恶性细胞,因此端粒DNA功能的异常是引起细胞衰老和癌变的重要标志之一。
端粒酶卡扎尔体蛋白1(telomeraseCajalbodyproteinl,TCAB1)是2009年新发现的端粒酶关键核心蛋白组分,它能把端粒酶从卡扎尔体运输到端粒末端的端粒合成目的地,以确保端粒长度维持机制的正常启动。TCAB1表达的缺失虽然不会影响端粒酶逆转录酶(TERT)自身的活性剂对端粒酶RNA组分(TERC)的催化,但可在细胞的S期阻止端粒酶复合物运送至端粒末端合成位点,最终导致端粒的形成和维持受到限制,引起端粒变短,这也意味着癌细胞会更快地死亡。袁平等人(袁平.RNAi沉默TCAB1表达对肺腺癌A549细胞生物学行为的影响[D].浙江大学,2014)利用RNA干扰技术能显著抑制TCAB1mRNA和蛋白质的表达,沉默TCAB1的表达能抑制肺腺癌细胞A549细胞中端粒酶与端粒的结合,抑制癌细胞增殖,但是该研究结果显示RNAi沉默并不能引起癌细胞凋亡。
发明内容
本发明的目的在于通过设计、构建、筛选,最终提供一些基于CRISPR/Cas9系统,同时靶向人TCAB1基因的高效gRNA及其靶位点序列,并用其抑制人TCAB1基因的表达,抑制肿瘤细胞的增殖,引起癌细胞的凋亡。
为实现上述目的,本发明以CRISPR/Cas9系统原理及其gRNA的设计原理为基础,软件设计预测出六个靶位点,并以px458为表达载体,构建了gRNA/Cas9表达系统,并在肿瘤细胞模型中加以应用。通过筛选和一系列的分析测试,最终筛选出两条位于第2外显子上的有效靶点,针对这两个靶点设计的gRNA能使目标靶位点发生移码突变。在人肝癌细胞株(HepG2)中利用这两个靶点指导的CRISPR/Cas9系统,可以有效的敲除人TCAB1基因,这一系统易于操作,人TCAB1基因敲除效率高。本发明提供的gRNA指导的CRISPR/Cas9系统能高效、靶向阻断人TCAB1基因,对于CRISPR/Cas9系统充分发挥作用和肿瘤治疗的靶标研究有极其重要的作用。
本发明申请的技术方案如下:
1、6条靶向人TCAB1基因的高效gRNA设计合成以及gRNA/cas9表达系统构建。
2、在肿瘤细胞模型中分析检测gRNA对于肿瘤细胞增殖的抑制作用以及引起癌细胞的凋亡作用,筛选到两个有效的靶位点。
3、在肿瘤细胞模型中分析检测本发明gRNA指导的CRISPR/Cas9系统对于肿瘤细胞增殖的抑制作用以及引起肿瘤细胞凋亡的作用。
附图说明
附图1为T7EndonucleaseI酶切结果(其中1泳道为对照组,2泳道为实验组);
附图2为细胞克隆结果图;
附图3为测序结果图;
附图4为Western-blot检测TCAB1蛋白表达结果图;
附图5为流式检测细胞凋亡图(其中:A为对照组;B为实验组)。
具体实施方式
实施例1靶向人TCAB1基因的gRNA合成及载体构建
1、靶向人TCAB1基因的gRNA的选择和设计
在Genebank中找到人TCAB1基因的序列,在人TCAB1基因的外显子区域设计潜在靶位点。通过在线设计工具(http://crispr.mit.edu/)及gRNA的设计原则,评估人TCAB1基因序列上得分较高的靶位点设计gRNA,靶位点序列为SEQIDNO.1-SEQIDNO.6,并设计对应的寡核苷酸。
2、靶向人TCAB1基因的gRNA寡核苷酸序列的合成和真核表达载体的构建
将pSpCas9(BB)-2A-GFP(PX458)质粒(AddgeneplasmidID:48138,以下简称pSpCas9(BB)),用BbSI酶切,37℃水浴1小时后,1%的琼脂糖电泳,回收酶切产物(TAKARA胶回收试剂盒)。
酶切体系如下:
将靶位点序列为SEQIDNO.1的两寡核苷酸退火,形成带有粘性末端的短双链DNA,反应体系如下:
将上述反应体系在200ulPCR管中混合均匀,然后将PCR管在37℃水浴锅中处理30min,再放入500ml沸水中,自然冷却至室温。
连接体系:
将带有粘性末端的双链短DNA产物连入酶切后的pSpCas9(BB)线性片段,将连接产物转化大肠杆菌DH5α感受态细胞(TakaraCode:D9057A),并涂布于Ampicillin浓度为100μg/ml的LB固体平板上培养过夜,挑取生长良好的单克隆,于15mlAmpicillin浓度为100μg/ml的LB液体培养基中,37℃振荡培养过夜,提取质粒,命名为px458-TCAB1-1。
3、无内毒素质粒DNA的制备
A、取px458-TCAB1-1质粒1μL加入100μLDH5α感受态细胞中吹匀,冰中静置20min,再放入42℃水浴90s,迅速置于冰浴中3min,加入500μLLB液体培养基,放置摇床180rpm37℃1h,取菌液100μL均匀涂布于Ampicillin浓度为100μg/ml的LB固体培养基37℃培养过夜。
B、取单菌落于3mlAmpicillin浓度为100μg/ml的LB液体培养基中,250rpm、37℃振荡培养8小时;从中取300μL菌液接种于300mlAmpicillin浓度为100μg/ml的LB液体培养基中,并于250rpm、37℃振荡培养12~16小时;
C、收集菌液,然后在4℃、4000rpm条件下离心15min,弃上清,收集菌体,然后按照QIAGENEndoFreePlasmidMaxiKit试剂盒说明书操作步骤提取质粒,得无内毒素的px458-TCAB1-1质粒。
实施例2脂转染HepG2细胞
转染前3天,复苏人肝癌细胞(HepG2细胞,中科院上海细胞库),将细胞放入加有10%的FBS+DMEM培养瓶中,于37℃、5%CO2的培养箱中培养,转染前一天,传代培养复苏细胞。
将培养HepG2细胞T75瓶中的培养基吸净,加入2ml4℃冰箱取出的0.25%胰酶,使其均匀覆盖瓶底,置于37℃培养箱中3-5min,取出,摇晃可发现细胞于底部脱离,将其全部晃下,加入3ml37℃水浴中预热的10%DMEM,用10ml移液管进行吹打,吹打6-8次,不留死角,瓶口处较难吹打可将移液管对准培口,小力将培养基打出即可覆盖到接近瓶口的细胞。之后,将所有细胞吸出,置于15ml离心管中,取50ul混匀后的细胞于1.5mleppendorf管中,加入450ul10%DMEM,即为10倍稀释,混匀,取10ul细胞于计数板中计数。传代当天记为第一天,若第二天进行转染,铺900-1000万/T75;若第三天转染,铺350-400万/T75。每瓶T75加10ml10%DMEM培养基。转染当天观察细胞密度,80-90%满即可进行转染。
采用脂质体转染法将px458-TCAB1-1转染HepG2细胞。转染体系及试剂使用LipofectamineTM2000(invitrogen公司),转染详细步骤参照转染说明书。
转染48小时后,利用胰酶消化转染后贴壁的细胞,离心收集细胞,吸掉废液加入1mlPBS重悬细胞,取500ul放入原瓶中继续培养,剩余细胞放入1.5ml离心管,提取DNA(按照DNA提取试剂盒说明书进行)。
以提取的DNA为模板(未转染细胞DNA为对照组),扩增靶点序列。PCR反应体系如下:
PCR扩增程序:95℃预变性3min;95℃变性30s,58℃退火30s,72℃延伸40s,30个循环后72℃延伸5min,最后4℃保温。
PCR产物用T7EndonucleaseI50℃水浴酶切1h,酶切体系如下:
T7EndonucleaseI酶切结果如图1所示,结果显示靶位点序列为SEQIDNO.1的靶点序列发生突变,具有高活性。
将剩余贴壁的细胞用胰酶消化,离心吸掉废液,加入1mlPBS重悬细胞,采用梯度稀释的方法,将细胞放入10cm培养皿中培养,培养8-10天时可观察到细胞克隆比较明显,每个集簇细胞数量约在500-1000个,如附图2所示。
将细胞消化,移至96孔板中培养,传代至6孔板时,消化细胞,离心重悬,取一半提取DNA鉴定,另一半细胞继续培养。
实施例3PCR产物克隆送样测序检测
按照实施例2的方法进行PCR反应,PCR产物用TAKARA试剂盒进行纯化后连接至PMD18-T载体上,连接体系为:
16℃下连接2小时。取感受态细胞DH5α,放置冰中融化5min,加入10ul连接产物吹匀,放置冰中20min。42℃热击90s,迅速转入冰浴中静置3min,加入500ul的LB液体培养基,置于摇床中,37℃180rpm1h。取菌液100ul均匀涂布于LB固体培养基(含1/1000AMP),37℃培养过夜。
挑3个取单菌落,分别放入3mlLB液体培养基(含3ulAMP),37℃200rpm12h。以1ul菌液为模板进行PCR鉴定,均为阳性。将菌液送样到上海生工进行测序。
同理,验证靶位点序列为SEQIDNO.2-6的gRNA对目标靶位点发生移码突变的影响。结果显示靶位点序列为SEQIDNO.1-2的gRNA能使目标靶位点发生移码突变,其测序结果如图3所示。
实施例4Western-blot检测TCAB1蛋白表达情况
1.蛋白检测样本的制备
取处于对数生长期的HepG2细胞,按3ml完全培养基/孔的量接种于6孔板细胞培养皿中,1×105细胞/孔。设计TCAB1敲除组和阴性对照组,置于细胞培养箱内贴壁过夜。随后PBS清洗细胞3次。每个培养皿中加入200ulRIPA裂解液,细胞刮刀刮取细胞集中于裂解液内,将样品转移至1.5ml离心管。样品置于冰上充分裂解30min,期间每隔10min在振荡器上振荡10秒。裂解结束后,4℃,12000r/min离心5min,去除基因组DNA。将上清转移至新的1.5ml离心管中,加入等体积的2×SDS-PAGE上样缓冲液混合,100℃煮沸5min使蛋白充分变性。
2.蛋白样本SDS-PAGE电泳
①用蒸馏水冲洗玻璃板子及梳子和胶条,晾干。玻璃板及胶条对齐后放入夹中卡紧,然后垂直卡在架子上准备灌胶。
②配10%分离胶,摇匀后灌胶约5cm高度,胶上加一水封闭,约半小时后,胶充分凝固就可倒去胶上层的水并用吸水纸吸干。
③配5%浓缩胶,摇匀后灌胶,将剩余空间灌满浓缩胶,然后将梳子插入浓缩胶中。待浓缩胶凝固后,两手分别捏住梳子的两边竖直向上将其拔出。
④撤掉梳子后置于电泳槽中,灌入电泳缓冲液并赶走上样孔中的气泡,每个孔上样30ug,加蛋白Marker5ul。
⑤电泳。
3.蛋白质的转移
将切好的与凝胶面积大小相同的PVDF膜先在甲醇中浸泡1-2min。在加有转膜液的托盘里放入转膜用的夹子,海绵垫、玻棒、滤纸和甲醇浸过的PVDF膜。将夹子打开使黑的一面保持水平。在上面垫一张海绵垫,用玻棒来回擀几遍以擀走里面的气泡。在垫子上垫三层滤纸,擀去气泡。
将凝胶从电泳槽中取出,小心剥下分离胶盖于滤纸上,将膜盖于胶上,再盖3张滤纸并除去气泡,最后盖上另一个海绵垫,合起夹子。将夹子放入转移槽中,电泳装置冰浴,250mA,转膜120min。
4.封闭和抗体结合反应
转膜完毕后,用3%BSA封闭液室温封闭1小时。按抗体说明书用3%BSA封闭液稀释TCAB1抗体(美国SantaCruz公司)。4℃孵育过夜。根据目标蛋白的分子量,以蛋白Marker显示的各条带分子量为标准将PVDF膜剪开,分别放入含对应抗体稀释液的15ml离心管中,室温反应2小时。之后用TBST漂洗PVDF膜3次,每次10分钟。加入对应的HRP标记的羊抗鼠IgG(美国abcom公司,用3%BSA封闭液稀释1∶2000稀释),室温反应1小时。TBST漂洗PVDF膜3次,每次10分钟。
5.显影
将膜在ECL溶液(1mlSolutionA+1mlSolutionB)中避光浸泡3min后压片、曝光、显色(Bio-Rad凝胶成像系统进行图像采集)。
实验结果显示(图4),通过Westernblot对转染后HepG2细胞中TCAB1蛋白质表达进行检测,TCAB1基因敲除组与对照组相比,其TCAB1蛋白几乎没有表达。
实施例5细胞凋亡检测
按照实施例2的方法培养细胞。把细胞培养液吸出至1.5ml的离心管内,PBS洗涤贴壁细胞一次,加入适量胰酶细胞消化液消化细胞。室温孵育至轻轻吹打可以使贴壁细胞吹打下来时,吸除胰酶细胞消化液。加入细胞培养液,稍混匀,转移到离心管内,1000rpm离心5min,弃上清液,收集细胞,用PBS吹打重悬细胞并计数。取5-10万细胞,1000rpm离心5min,弃上清液,加入195ulAnneixinV-FITC(碧云天)结合液轻轻重悬细胞。加入5ulAnnexinV-FITC,轻轻混匀。室温避光孵育10min,1000rpm离心5min弃上清,加入190ulAnnexinV-FITC结合液轻轻重悬细胞。加入10ul碘化丙啶液,轻轻混匀,冰浴避光放置。
流式细胞仪检测细胞凋亡情况,检测结果如图4所示,其中,左下象限代表正常细胞,左上象限代表细胞手机收集过程中的损伤细胞,右下象限代表早期凋亡细胞,右上象限为晚期凋亡细胞和坏死细胞,右上象限和右下象限想家得到总的凋亡细胞比率。流式检测显示:SEQIDNO.1-2靶位点序列的两条gRNA指导的CRISPR/Cas9系统,可以有效的敲除人TCAB1基因,基因敲除TCAB1基因细胞实验组的细胞凋亡水平明显高于未敲除TCAB1基因细胞对照组的细胞凋亡水平。
Claims (5)
1.在CRISPR/Cas9特异性敲除人TCAB1基因中用于特异性靶向人TCAB1基因的gRNA,所述gRNA在人TCAB1基因上的靶序列符合5’-N(20)-NGG3’或者5’-CCN-N(20)-3’的序列排列规则,在人TCAB1基因上的靶序列是唯一的,其特征在于:所述gRNA在人TCAB1基因的靶向位点位于人TCAB1基因的第2外显子上。
2.根据权利要求1所述的在CRISPR/Cas9特异性敲除人TCAB1基因中用于特异性靶向人TCAB1基因的gRNA,其特征在于:所述靶向位点如序列表SEQIDNO.1-2任意一条序列所示。
3.根据权利要求1-2所述的在CRISPR/Cas9特异性敲除人TCAB1基因中用于特异性靶向人TCAB1基因的gRNA,其特征在于:所述gRNA指导的CRISPR/Cas9系统能促进肿瘤细胞的凋亡。
4.根据权利要求3所述的在CRISPR/Cas9特异性敲除人TCAB1基因中用于特异性靶向人TCAB1基因的gRNA,其特征在于:所述的肿瘤细胞为人肝癌细胞。
5.根据权利要求1-2所述的在CRISPR/Cas9特异性敲除人TCAB1基因中用于特异性靶向人TCAB1基因的gRNA,其特征在于:所述gRNA指导的CRISPR/Cas9系统在治疗肿瘤的新型药物中的应用。
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