CN108148835A - CRISPR-Cas9靶向敲除SLC30A1基因及其特异性的sgRNA - Google Patents

CRISPR-Cas9靶向敲除SLC30A1基因及其特异性的sgRNA Download PDF

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CN108148835A
CN108148835A CN201711282037.8A CN201711282037A CN108148835A CN 108148835 A CN108148835 A CN 108148835A CN 201711282037 A CN201711282037 A CN 201711282037A CN 108148835 A CN108148835 A CN 108148835A
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slc30a1
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马佩敏
孙子豪
杨兴林
潘讴东
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Yuan Biotechnology (shanghai) Ltd By Share Ltd
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Abstract

本发明公开了CRISPR/Cas9靶向敲除人乳腺癌细胞SLC30A1基因及其特异性sgRNA。首先是获得特异性靶向SLC30A1基因的sgRNA，其碱基序列如SEQ ID NO.1所示；其次是构建SLC30A1基因的sgRNA到慢病毒载体***，该***含有Cas9蛋白；最后将含有该sgRNA的CRISPR/Cas9慢病毒感染人乳腺癌细胞MDA‑MB‑231，获得SLC30A1蛋白表达水平显著降低的细胞株。本发明具有操作步骤简单、sgRNA靶向性好、且对SLC30A1基因切割效率高；此外，所构建的CRISPR/Cas9慢病毒***具有敲除效率高的优势，并能特异性敲除SLC30A1基因，得到敲除SLC30A1基因的人乳腺癌细胞，从而为进一步研究乳腺癌细胞中SLC30A1的作用机制提供强有力的工具。

Description

CRISPR-Cas9靶向敲除SLC30A1基因及其特异性的sgRNA
技术领域
本发明属于基因工程领域，更具体地说，涉及CRISPR-Cas9 特异性敲除人SLC30A1基因的方法以及用于特异性靶向SLC30A1基因的sgRNA。
背景技术
SLC30A1(锌运转体)成员1是一种蛋白质在人类由SLC30A1编码基因。必需微量元素锌通过催化和结构作用参与机体多种酶和蛋白功能,与机体发育、脑功能、骨骼生长、生殖健康及免疫功能等密切相关。补充锌可以一定程度防治儿童腹泻、慢性丙型肝炎、急性下呼吸道感染以及感冒等疾病,然而过多的锌具有毒性。因此,机体存在复杂的锌离子稳态体系维持锌离子的吸收、储存和丢失的平衡过程。SLC30A家族又称ZnT家族,共有10个成员,多个家族成员可协助锌离子从细胞质内流出到细胞外或流进到细胞器内。研究提示ZnT1、ZIP4和ZIP5参与小肠锌离子吸收过程,ZIP10和ZnT1参与肾脏锌离子再吸收过程,ZIP5、ZnT2和ZnT1参与胰腺锌离子分泌丢失过程。另有证据证明SLC30A家族的蛋白还可能参与许多疾病包括肿瘤及糖尿病的发生和发展。
乳腺癌是威胁女性生命健康的一大疾病,探讨其发病机制寻找有效预防和治疗方法尤为重要。锌在乳腺癌中水平异常增高,其特异性跨膜转运蛋白锌转运体又和金属基质蛋白酶这一已知的肿瘤相关蛋白有相似结构,加之LIV-1***调节基因中的两个锌转运体成员ZIP6和ZIP10与乳腺癌的转移有很密切的关联,因此,探讨***受体性状不同的乳腺癌细胞MDA-MB-231中锌转运体的表达以及锌对锌转运体表达的影响很有必要。
CRISPR/Cas9是近几年发现的由RNA向导的Cas9核酸酶靶向目的基因进行编辑的新兴技术。CRISPR/Cas9是最先在古细菌中发现的，是研究者发现古细菌对外来生物信息不断攻击而演化来的一种获得性防御机制在CRISPR/Cas9***中，crRNA( CRISPR-derivedRNA )通过DNA碱基配对原理与tracrRNA( trans-activating RNA )结合形成双链RNA，指导Cas9蛋白在crRNA向导的DNA序列靶定目的位点切断双链DNA。相比于传统的锌指核酸酶(ZFNs)技术，转录激活样效应因子核酸酶（TALENs）技术等，CRISPR-Cas 技术具有独特的优势：设计更简单，特异性更高，速度快、生殖系转移能力强及简单经济的特点。
参考文献：
1)Moonhuhn Ryu, Louis A. Lichten, Juan P. Liuzzi, et al. ZincTransporters ZnT1 (Slc30a1), Zip8 (Slc39a8), and Zip10 (Slc39a10) in MouseRed Blood Cells Are Differentially Regulated during Erythroid Development andby Dietary Zinc Deficiency[J]. Journal of Nutrition, 2008, 138(11):2076-2083.
2)Fernandez E L, Dencker L, Tallkvist J. Expression of ZnT-1 (Slc30a1)and MT-1 (Mt1) in the conceptus of cadmium treated mice[J]. ReproductiveToxicology, 2007, 24(3–4):353-358.
3)Muraina, Issa. Reverse genetics analysis of biological functions ofzinc transporters Znt1 (Slc30a1) & Zip10 (Slc39a10) in zebrafish[J]. KingsCollege London, 2013。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种靶向敲除SLC30A1基因的sgRNA。
本发明的另一目的在于提供所述一种靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***的应用。
本发明的另一目的在于提供靶向敲除SLC30A1基因的人肠癌细胞MDA-MB-231细胞的应用。
本发明的目的通过下述技术方案实现：一种靶向敲除SLC30A1基因的sgRNA，选自DNA序列如下的SLC30A1sgRNA：
SLC30A1sgRNA的序列如下：
SLC30A1sgRNAoligo1：5’-cacc GCTCTTAACGCGAGGCCCCT-3’；
SLC30A1sgRNAoligo2：5’-aaac AGGGGCCTCGCGTTAAGAGC-3’；
一种靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***，含有上述靶向敲除SLC30A1基因的sgRNA的DNA序列。
所述的靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***的构建，包括如下步骤：
（1）使用BsmBI酶切CRISPR/Cas9慢病毒载体LentiCRISPRV2，得到酶切后的CRISPR/Cas9慢病毒载体；
（2）将上述靶向敲除SLC30A1基因的sgRNA的DNA序列磷酸化后与酶切后的CRISPR/Cas9慢病毒载体连接，得到靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***。
步骤( 2 )中所述的DNA序列是将寡核苷酸链1( oligo1 ) 和寡核苷酸链2(oligo2 )退火得到双链序列。
所述的靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***在制备敲除RITA基因的细胞株中的应用。
一种敲除SLC30A1基因的细胞株，是将所述的靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***转染目的细胞株得到的。
所述的敲除SLC30A1基因的细胞株，具体是通过如下步骤构建得到：
1）将所述的靶向敲除SLC30A1基因的CRISPR/Cas9慢病毒***通过包装细胞进行包装，得到慢病毒颗粒；
2）将慢病毒颗粒感染目的细胞株，得到敲除SLC30A1基因的细胞株。
所述的目的细胞株优选为肿瘤细胞株。
所述的肿瘤细胞株优选为乳腺癌细胞株。
所述的乳腺癌细胞株优选为人乳腺癌癌永生化细胞MDA-MB-231。
所述的敲除SLC30A1基因的细胞株，是MDA-MB-231细胞中的SLC30A1基因缺失或***核苷酸得到的细胞株。
本发明相对于现有技术具有如下的优点及效果：
本发明提供SLC30A1基因的sgRNA能有效靶向SLC30A1基因, 将其构建入CRISPR/Cas9慢病毒***，该***能敲除SLC30A1基因，得到敲除SLC30A1基因的细胞株，从而有利于研究细胞株中的SLC30A1的作用机制。
附图说明
图1 为本发明实施例中使用的载体质粒lentiCRISPR v2 的质粒图谱；
图2 为本发明实施例中使用的表达载体质粒lentiCRISPRv2-hSLC30A1sg 的质粒图谱；
图3 为本发明实施例中使用的包装质粒pLP-VSVG 的质粒图谱；
图4 为本发明实施例中使用的包装质粒psPAX2 的质粒图谱；
图5 为MDA-MB-231细胞病毒感染含有SLC30A1sgRNA的活性鉴定测序图；
图6 为敲除SLC30A1基因的单克隆MDA-MB-231细胞株1中SLC30A1基因的测序图；
图7 为敲除SLC30A1基因的单克隆MDA-MB-231细胞株2中SLC30A1基因的测序图；
图8 为敲除SLC30A1基因的单克隆MDA-MB-231细胞株3中SLC30A1基因的测序图；
图9 为敲除SLC30A1基因的单克隆MDA-MB-231细胞株4中SLC30A1基因的测序图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
1 .使用CRISPR/Cas9技术构建敲除SLC30A1质粒
1 .1sgRNA寡核苷酸链合成.
使用CRISPR在线设计工具( http://crispr .mit .edu/ )根据评分***，在SLC30A1的外显子2上设计1个20bp的sgRNA，并通过BLAST验证无非特异性基因。编码链模板5′端添加CACC，非编码链模板3′端添加AAAC，与BsmBI酶切后形成的粘性末端互补，设计1对CRISPR寡核苷酸链，见表1 SLC30A1靶向位点及sgRNA寡核苷酸序列
1 .2载体构建
1.2.1使用BsmBI酶切2μg lentiCRISPRv2质粒(购于Addgene公司)，2h，37℃.
1.2.2使用GENRY胶回收试剂盒纯化酶切质粒产物，按说明书进行操作
1.2.3磷酸化并退火sgRNAoligos：
PCR仪器退火程序：37℃ 30min，95℃维持5min，每分钟降低5℃至25℃，4℃维持。
1.2.4将退火形成的oligo双链和酶切后的lentiCRISPRv2载体直接连接，室温下，10min。
1.2.5将连接后的质粒转化至感受态细胞DH5α中，均匀涂至LB固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。
1 .3挑取单个菌落扩大培养并质粒小提。
1.4测序鉴定质粒构建成功，并命名为lentiCRISPRv2-hSLC30A1sg。
1.5敲除效率验证
用含10％胎牛血清的高糖DMEM培养基于5％CO2，37℃恒温培养293T细胞(购于美国ATCC细胞库)。取对数期细胞以1×105/孔接种到24孔板培养。待细胞融合度达到70％～8 0％ 时更换成Opti-MEM培养基，1 小时后将相应的CRISPR/Cas9敲除质粒0.8μg 经Lipo2000试剂，转染到293T细胞中，转染48小时候，荧光显微镜下观察转染效果，消化收集细胞，采用基因组DNA提取试剂盒提取细胞基因组DNA;以各组细胞基因组DNA为模板，利用表2的引物PCR扩增靶向序列。用SLC30A1-4-1和SLC30A1-4-2进行PCR,得到904bp的片段a(高GC)，以a为模板，用SLC30A1-4-3和SLC30A1-4-4进行PCR，得到400bp的片段b(高GC)，该片段电泳后胶回收用于测序引物SLC30A1-4-3,表2
2 .包装慢病毒
100mm 皿种入293TN细胞4X106，培养24h( 37℃ ,5％CO2 )后，更换新鲜培养基，转染包装细胞：
a .混匀以下3个质粒：
包装质粒psPAX2 12μg
辅助质粒pLP-VSVG 10μg
表达质粒lentiCRISPRv2- hSLC30A1sg 22μg
加入CaCl2 250μl
加入ddH2O至总体积为500μl，37℃放置20min；
b .2 X BES 500μl，37℃放置20min；
c .把a中500μl三质粒混合液逐滴加入b中，并轻轻混匀；
d.小心的将c中混合液加入种有293TN的100mm 皿中；培养12h后移除感染液，更换10ml新鲜培养基生产病毒液；培养48h后，收获病毒液至50ml离心管中，并重新向100mm 皿中补加10ml培养基再次生产病毒；培养24h后，再次收获病毒液，室温离心，1000rpm，10min；使用0.22μm的滤器过滤病毒上清液；将病毒上清液装入超离管中，4℃，100000g，2h；弃去上清液后用Opti-MEM重悬过夜；再次使用0.22μm的滤器过滤重悬的病毒；分装后，-80℃保存。
3 .病毒感染
1 )将要感染的MDA-MB-231细胞接种到六孔板中，过夜，待细胞融合度约为50％时，移去培养基，更换2ml新鲜培养基；
2 )每孔加入50μl病毒液，再加入12μl聚凝胺polybrene，至终浓度为6μg/ml；
3 )37℃培养12h后，移除培养液，更换新鲜培养液后继续培养48h。
4 .筛选稳定细胞株
感染结束48h后，向每孔加入嘌呤霉素( 2 .0μg/ml )，隔天换液，并保持培养基的嘌呤霉素浓度恒定，筛选阳性克隆细胞，所得细胞株命名为MDA-MB-231-SLC30A1，并使用有限稀释法挑选MDA-MB-231-SLC30A1单克隆敲除细胞株。
5 .稳定细胞株鉴定
提取各组敲除SLC30A1的单克隆细胞株基因组DNA测序，鉴定出sgRNA是有活性的（图5），并且找到4种类型的具有SLC30A1基因缺失或***突变的稳定细胞株(图6-9 )。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 和元生物技术(上海)股份有限公司
<120> CRISPR-Cas9 靶向敲除SLC30A1基因及其特异性的sgRNA
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<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial synthesis)
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Claims (5)

1.一种CRISPR/Cas9 靶向敲除人乳腺癌细胞SLC30A1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SLC30A1 基因的sgRNA；其次构建SLC30A1基因的sgRNA到慢病毒载体***；然后将此慢病毒载体***感染人乳腺癌细胞MDA-MB-231，得到的SLC30A1基因敲除细胞株。
2. 根据权利要求1所述的CRISPR/Cas9 靶向敲除人乳腺癌细胞SLC30A1基因及其特异性的sgRNA，其特征在于包括如下步骤：
(1) 提供sgRNA，所述sgRNA 在SLC30A1基因上的靶序列符合5’-N(19)G 的序列排列规则，所述sgRNA 在SLC30A1基因上的靶序列位于基因的外显子，所述sgRNA 在SLC30A1基因上的靶序列位于不同的各种剪切形式的共有外显子上，所述sgRNA 在SLC30A1基因上的靶序列是唯一的，并且所述sgRNA 在SLC30A1 上的靶位点序列如序列表SEQ ID NO.1序列所示，在所述sgRNA 在SLC30A1 上的靶位点序列的5’加上CACC, 合成得到正向寡核苷酸即Forward oligo ；获得sgRNA 在SLC30A1上的靶位点序列的互补链，并且在互补链的5’加上AAAC 合成得到反向寡核苷酸即Reverse oligo ；将合成的1 对互补的sgRNA 寡聚核苷酸的forward oligo 和reverse oligo 成对变性、退火，退火之后形成可以连入U6 真核表达载体的双链sgRNA寡聚核苷酸；
(2) 线性化序列如序列表SEQ ID NO.10 所示的lentiCRISPRv2质粒；将退火的双链sgRNA 寡聚核苷酸与线性化的携带Cas9 基因的表达载体lentiCRISPRv2连接获得携带含相应靶序列的sgRNA 寡聚核苷酸和Cas9 基因的表达载体lentiCRISPRv2-h SLC30A1sg 质粒，转化感受态细菌并涂Amp+ 平板，挑取单克隆并用序列如序列表SEQ ID NO.9 所示的通用引物U6 通过测序鉴定出阳性克隆，并对所述阳性克隆摇菌、提取质粒；
（3）用所述携带有sgRNA 寡聚核苷酸和Cas9 基因的表达载体lentiCRISPRv2-hSLC30A1sg 质粒、序列为SEQ ID NO.11和SEQ ID NO.12 的pVSVg (AddGene 8454) andpsPAX2 (AddGene 12260)包装质粒和包装细胞系包装出同时携带靶向DEAF1基因的sgRNA和Cas9 的假型慢病毒；
（4）使用所述假型慢病毒感染目的细胞，并进一步培养；然后收集被感染的目的细胞，以其基因组DNA 为模板扩增包含所述靶序列的基因片段，T7EN1 酶切检测和TA 克隆测序确认SLC30A1 基因已经被敲除并获得基因敲除的细胞。
3.根据权利要求1 所述的在CRISPR-Cas9 特异性敲除人SLC30A1基因的方法中用到的重组表达载体lentiCRISPR v2- SLC30A1，其特征在于，所述重组表达载体的骨架载体的序列如序列表中SEQ ID NO ：10 所示。
4.根据权利要求1所述的CRISPR-Cas9 特异性敲除人SLC30A1基因的方法，其特征在于，所述包装质粒为pVSVg (AddGene 8454) and psPAX2 (AddGene 12260) ；所述包装细胞系为HEK293TN 细胞。
5.根据权利要求1所述的CRISPR-Cas9 特异性敲除人SLC30A1 基因的方法，其特征在于，所述目的细胞为人MDA-MB-231细胞。
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