CN111733160A - 使用CRISPR-Cas9***对间充质干细胞进行CKIP-1基因敲除的方法 - Google Patents
使用CRISPR-Cas9***对间充质干细胞进行CKIP-1基因敲除的方法 Download PDFInfo
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Abstract
本发明提供了一种采用CRISPR‑Cas9***对间充质干细胞进行CKIP‑1基因编辑,特别是涉及一种构建CKIP‑1基因敲除的人骨髓间充质干细胞细胞系的建立,用于骨组织生理功能以及创伤修复再生后续研究。其中构建并获得了一个特异的gRNA,能够显著提高间充质干细胞内CRISPR‑Cas9针对CKIP‑1的基因编辑效率。本发明构建的CKIP‑1基因敲除是在基因组水平上形成移码突变,故可以随着细胞***、增殖传递到下一代,形成稳定的CKIP‑1基因敲除细胞系。
Description
技术领域
本发明涉及基因工程技术领域,具体的涉及一种利用CRISPR-Cas9技术敲除间充质干细胞CKIP-1基因的方法。
背景技术
间充质干细胞(mesenchymal stem cell,MSC),又称间充质基质细胞(mesenchymal stromal cells),源于发育早期的中胚层和外胚层,几乎存在于所有生物组织类型中,如骨髓、脂肪组织和牙髓等。MSC是具有自我更新能力和分化为多种细胞类型的多能干细胞,包括成骨细胞、脂肪细胞、肌细胞、肝细胞、类固醇生成细胞、骨骼肌细胞、平滑肌细胞、运动神经元和内皮细胞等。同时能分泌产生大量细胞因子和生长因子等生物活性因子,以旁分泌方式作用于微环境,发挥促血管生成、抗纤维化、抗凋亡和免疫调节功能。此外,MSC备显著归巢能力,可迁移至损伤部位进行免疫调节、位点特异性分化和支持造血等。更为重要的是,不论是自体还是同种异源的MSC,一般都不会引起宿主的免疫反应。这些特性使MSC成为细胞治疗的优秀候选细胞,广泛用于实验、临床研究和基因工程中,用于骨骼、心脏、软骨、中枢神经、皮肤等的再生,在组织修复领域具有广阔的应用前景。
基因编辑技术主要研究基因相关功能与表型的关系,已成为现代分子生物学的研究热点,未来期望被用于人类遗传性疾病、癌症、自身免疫疾病等重大疾病的治疗。CRISPR/Cas***广泛存在于细菌及古生菌中,在RNA指导下可以降解病毒或噬菌体等外源DNA。CRISPR-Cas***分为Ⅰ、Ⅱ、Ⅲ型,其中Ⅱ型***中Cas9核酸内切酶Rnase将前体crRNA剪切为成熟crRNA,成熟crRNA含有与目标DNA互补配对的非重复间隔序列,通过识别PAM序列引导Cas9蛋白对目标DNA序列进行切割。现代常用的基因编辑技术CRISPR-Cas9就是由此改造成的,把人工设计的引导RNA(gRNA)与Cas9构成重组质粒,gRNA引导Cas9核酸酶对靶标基因组进行结合与切割,使DNA双链发生断裂,断裂的双链DNA被细胞内自然存在的DNA修复***修复,从而完成对靶标DNA的突变修饰。CRISPR-Cas9技术能够实现基因定点敲除、敲入和突变等基因编辑功能,目前已经被广泛应用于各类细胞和模式动物的基因组编辑,在疾病治疗方面取得了较大的突破。
酪蛋白激酶2-相互作用蛋白1(Casein kinase 2-interacting protein-1,CKIP-1)是近年发现的负调控骨形成的重要因子。资料显示,糖皮质激素诱导的骨质疏松大鼠成骨细胞及体外糖皮质激素处理成骨细胞后CKIP-1的表达均显著增加(Liu J,Lu C,Wu X,etal.Targeting osteoblastic casein kinase-2interacting protein-1to enhanceSmad-dependent BMP signaling and reverse bone formation reduction inglucocorticoid-induced osteoporosis[J].Scientific Reports,2017,7:41295);靶向沉默CKIP-1后促进健康和骨质疏松小鼠的骨形成(Guo B,Zhang B,Zheng L,etal.Therapeutic RNA interference targeting CKIP-1with a cross-species sequenceto stimulate bone formation.Bone.2014;59:76-88)。SiCKIP-1可以使BMSCs成骨及成脂分化能力增强(Jia S,Yang X,Song W,et al.Incorporation of osteogenic andangiogenic small interfering RNAs into chitosan sponge for bone tissueengineering.Int J Nanomedicine.2014;9:5307-5316)。由此可见,CKIP-1可能作为一个新的骨缺损修复靶点,通过敲除这个基因有可能使MSCs的成骨分化能力增强。另外基于MSCs的多能性,为了研究CKIP-1在成骨分化中的作用,加深对骨代谢及骨重建的理解,建立敲除CKIP-1的MSCs变得尤为重要。
研究内容
本发明的目的是提供一种利用CRISPR-Cas9技术敲除MSCs细胞CKIP-1基因的方法。
本发明的另一个目的是提供利用上述方法制备CKIP-1基因敲除的MSCs细胞系,以其作为组织工程种子细胞在研究维持骨组织生理功能以及创伤修复再生中的应用。
为实现上述目的,本发明首先提供一种CRISPR-Cas9***的靶标,根据人CKIP-1基因序列的6号外显子,设计的具体可选择的靶位点如下(划线部分表示PAM基序):
CKIP1-sgRNA1:5’to 3’TGAGAGCTTTCGGGTTGACCTGG
CKIP1-sgRNA2:5’to 3’GGTCTGAGTCCGCTCTCCGCCGG
CKIP1-sgRNA3:5’to 3’ACTGCTTGCCCGGTCTGCGGAGG
CKIP1-sgRNA4:5’to 3’AAGCAGTCTCTCCCGACCTTGGG
CKIP1-sgRNA5:5’to 3’CAGAATCCGGCGGAGACCGAGGG
CKIP1-sgRNA6:5’to 3’CCGGTACTGACTGTGCGGGGTGG
本发明还提供一种CKIP-1基因打靶载体,所述打靶载体是基于CRISPR-Cas9***的gRNA表达载体。
用于构建所述CRISPR-Cas9***的gRNA表达载体的出发载体为pX458,是将gRNA作用位点的DNA序列5'端加上CACC得到正向寡核苷酸序列,在其互补链的5'端加上AAAC得到反向寡核苷酸序列。将两条互补序列退火形成的双链DNA与经过BbsI酶切的pX458载体连接构建得到的。
利用打靶载体转染MSCs细胞,获得编辑阳性细胞,PCR测序鉴定。
用于鉴定中靶阳性细胞克隆的特异性PCR引物序列如下:
CKIP-1.F:GACCTTGGACTTGATCCAAGAGGAAG
CKIP-1.R:GTGGAGTGGTCAGACTGTGTCTTTAC
用流式分选编辑阳性细胞,随后繁殖、收获所需干细胞。
本发明提供了一种构建CKIP-1基因敲除的间充质干细胞系方法,具有以下优点:
(一)利用CRISPR-Cas9***首次从MSCs细胞中敲除CKIP-1基因,简便、快速,可以作为理想的组织工程种子细胞。
(二)基于CRISPR-Cas9方法,与沉默、干扰、敲低等手段相比,能够更有效的敲除CKIP-1。
(三)构建CKIP-1敲除的MSCs,筛选并优化获得了最佳的sgRNA,敲除效率高,传代稳定。
附图说明
图1为pX458质粒图。
图2为pX458中sgRNA***位点图。
图3为pX458-sgRNA菌落PCR电泳鉴定图。泳道1和2:sgRNA1,泳道3和4:sgRNA2,泳道5和6:sgRNA3,泳道7和8:sgRNA4,泳道9和10:sgRNA5,泳道11和12:sgRNA6,泳道13:DL2000 Marker。
图4为sgRNA3活性验证测序图。
图5为流式分选敲除CKIP-1基因后的单克隆MSC细胞株中CKIP-1基因测序比对图。
具体实施方式
下面结合实施例及附图对本发明做进一步详细的描述,但本发明的实施方式不限于此。
下述实施例中所用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
pSpCas9(BB)-2A-GFP(PX458)质粒:Addgene公司,产品目录号48138。
人骨髓间充质干细胞:武汉普诺赛生命科技有限公司,产品货号CP-H166。
实施例1构建CRISPR表达载体
1.1靶向人CKIP1基因的gRNA的选择和设计
在Genebank上查找人CKIP1基因序列,在其6号外显子上设计靶位点,通过在线设计工具(http://crispr.mit.edu/)及gRNA的设计原则,寻找合适的靶点,序列如下:
CKIP1-sgRNA1:5’to 3’TGAGAGCTTTCGGGTTGACCTGG
CKIP1-sgRNA2:5’to 3’GGTCTGAGTCCGCTCTCCGCCGG
CKIP1-sgRNA3:5’to 3’ACTGCTTGCCCGGTCTGCGGAGG
CKIP1-sgRNA4:5’to 3’AAGCAGTCTCTCCCGACCTTGGG
CKIP1-sgRNA5:5’to 3’CAGAATCCGGCGGAGACCGAGGG
CKIP1-sgRNA6:5’to 3’CCGGTACTGACTGTGCGGGGTGG
根据上述的gRNA,在其5'端加上CACC得到正向寡核苷酸序列,在其互补链的5'端加上AAAC得到反向寡核苷酸序列,分别合成正向和反向寡核苷酸序列。正向:5’-CACCGNNNNNNNNNNNNNNNNNNNN反向:CNNNNNNNNNNNNNNNNNNNNCAAA-5’设计对应的寡核苷酸序列SEQ ID NO:3-SEQ ID NO:14(即sgRNA1-6)。
1.2靶向CKIP1基因的gRNA寡核苷酸序列的合成及真核表达载体的构建
1.2.1使用BbsI内切酶将pSpCas9(BB)-2A-GFP(PX458)质粒(Addgene plasmidID:48138,以下简称pX458)在37℃水浴条件下酶切1h,结束后进行1%琼脂糖凝胶电泳,采用DNA凝胶回收试剂盒(万类生物)回收线性化片段。酶切体系如下:
FD BbsI(FD BpiI) | 1μL |
10×FD buffer | 5μL |
pX458 | 10μL(500ng/μL) |
ddH<sub>2</sub>O | 34μL |
合计 | 50μL |
1.2.2将靶点序列为SEQ ID NO:3-SEQ ID NO:14对应的寡核苷酸链退火,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200μL PCR管内混匀,短暂离心至PCR管底,将PCR管放入PCR仪内按照以下程序进行缓慢退火:
37℃ | 30min |
95℃ | 5min |
95℃~22℃ | -1.5℃/min |
1.2.3将退火形成的Oligo双链和BbsI酶切后的线性化pX458在16℃条件下过夜连接,反应体系如下:
酶切回收产物 | 1μL |
退火产物 | 1μL |
T4 DNA Ligase | 1μL |
T4 Ligase buffer | 1μL |
ddH<sub>2</sub>O | 6μL |
总计 | 10μL |
1.2.4将连接后的产物转化至感受态大肠杆菌DH5α细胞中,均匀涂布至含有100μg/mL氨苄青霉素的LB固体培养基平板中,置于37℃培养箱中过夜培养,即可出现单菌落。
1.3挑取单菌落扩大培养并质粒小提
1.3.1准备若干个装有20μL无菌水的200μL无菌PCR管并标号,从上述过夜培养的培养基平板上挑取单菌落,在PCR管中用无菌水混匀,取2μL上述菌悬液转移至另一个空的无菌PCR管,并按照下表配制PCR反应体系,将所有组分混匀后短暂离心至管底:
菌悬液 | 2μL |
2×PCR master Mix | 12.5μL |
上游引物hU6F | 1μL |
下游引物Oligo(-) | 1μL |
ddH<sub>2</sub>O | 8.5μL |
总计 | 25μL |
将上述PCR反应体系放入PCR仪,按照以下程序进行PCR扩增:
1.3.2取上述PCR产物进行2%琼脂糖凝胶电泳验证,将扩增出目的条带的对应剩余菌悬液接种于含有100μg/mL氨苄青霉素为的LB液体培养基中,在200r/min、37℃条件下培养12-16h。
使用上述方法,共计得到6种无内毒素的基因编辑质粒。
实施例2建立CKIP-1基因敲除间充质干细胞模型
2.1对人骨髓间充质干细胞进行常规复苏和传代,以2×105个/孔接种至6孔培养板中,待细胞生长至60-70%融合度,采用6种质粒分别转染细胞,具体如下:
溶液1:Opti-MEM 100uL+lip3000 6μL,溶液2:Opti-MEM 100μL+DNA 2μg+p3000 5μL。将溶液1加到溶液2中,室温静置10-15min。
将上述混合液缓慢滴入细胞中,并不断轻轻震荡,然后将细胞放入37℃、5%CO2培养箱中培养,转染48h后加入胰酶消化贴壁细胞,离心收集,弃废液,加入1mL PBS缓冲液重新悬浮细胞,取500μL放入原六孔板中继续培养,剩余细胞放入1.5mL离心管,按照组织基因组DNA小量纯化试剂盒(万类生物)说明书进行操作,提取基因组DNA。
2.2基因组提取成功后在无菌的200μL PCR管中配制以下PCR体系,用于目的基因的扩增:
基因组DNA | 1μL |
2×PCR master Mix | 12.5μL |
上游引物CKIP-1.F | 1μL |
下游引物CKIP-1.R | 1μL |
ddH<sub>2</sub>O | 9.5μL |
总计 | 25μL |
将上述反应体系混匀,短暂离心至管底,放入PCR仪中,按照以下程序进行扩增:
2.3纯化PCR产物,测序,并对测序结果进行分析,结果见附图4。结果显示,SEQ IDNO:7对应的质粒起到了良好的基因敲除效果,测序峰图从第119个碱基(对应sgRNA的突变热点位置)开始出现杂乱的重叠峰,说明SEQ ID NO:7对应的sgRNA质粒对人CKIP-1具有较好的基因编辑效果。
2.4将基因编辑效果好的质粒转染的细胞稀释培养,使用流式细胞仪对带有GFP标记的细胞进行分选,计数,按每孔1个细胞接种至96孔培养板中,进行扩大培养,显微镜下观察荧光,并鉴定细胞克隆。提取细胞DNA并进行靶位点PCR,PCR产物连T载体,转化,挑取2个阳性克隆,提取质粒,送测序,结果见附图5。结果显示靶位点序列为SEQ ID NO:7的gRNA能使目标靶位点发生移码突变,可以建立稳定可靠的CKIP-1基因敲除MSCs模型。
最后所应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者同等替换,而不脱离本发明技术方案的精神和范围。
序列表
<110> 沈阳万类生物科技有限公司
<120> 使用CRISPR-Cas9***对间充质干细胞进行CKIP-1基因敲除的方法
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<170> SIPOSequenceListing 1.0
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Claims (7)
1.一种CRISPR-Cas9靶向敲除CKIP-1基因及其特异性的sgRNA,其特征在于,首先设计获得特异性靶向CKIP-1基因第6外显子的sgRNA;其次构建CKIP-1基因的sgRNA到表达载体***,然后将此载体***转染间充质干细胞,得到CKIP-1基因敲除细胞株。
2.根据权利要求1所述的特异性靶向CKIP-1基因第6外显子的sgRNA,其DNA序列如SEQID NO:7和SEQ ID NO:8所示,所述sgRNA在CKIP-1基因上的靶序列是唯一的。
3.根据权利要求2所述的特异性靶向CKIP-1基因第6外显子的sgRNA,其引物包括针对CKIP-1基因第6外显子上的引物对:
CKIP-1.F:如SEQ ID NO:1所示;
CKIP-1.R:如SEQ ID NO:2所示。
4.根据权利要求1所述的构建CKIP-1基因的sgRNA到表达载体***,是一种靶向敲除CKIP-1基因的CRISPR-Cas9重组表达载体pSpCas9(BB)-2A-GFP(PX458),其特征在于:含有特异性靶向CKIP-1基因第6外显子的sgRNA和Cas9蛋白的重组表达载体。
5.权利要求4所述的***,其特征在于:可以导入到基因编辑细胞中,筛选得到阳性细胞。
6.权利要求4的***在制备用于间充质干细胞基因编辑的试剂中的用途。
7.权利要求6所述的用途,其中间充质干细胞为人骨髓间充质干细胞(hMSCs)CP-H166。
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