CN107502608B - 用于敲除人ALDH2基因的sgRNA、ALDH2基因缺失细胞株的构建方法及应用 - Google Patents
用于敲除人ALDH2基因的sgRNA、ALDH2基因缺失细胞株的构建方法及应用 Download PDFInfo
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Abstract
本发明提供了一种用于敲除人ALDH2基因的sgRNA序列，所述sgRNA的靶DNA序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示序列中的至少一条。本发明还提供了一种敲除人肝癌细胞ALDH2基因的方法，为利用CRISPR/Cas系统在人肝癌细胞中对ALDH2基因进行改造。本发明还提供了两种ALDH2基因缺失细胞株细胞株，ALDH2参与机体重要的代谢功能，本发明提供的ALDH2基因敲除细胞株为外源性化学物或者外源性毒物在体内的代谢研究提供了有效的平台，对于慢性疾病(如酒精性肝脏疾病及糖尿病)及肿瘤相关疾病的研究提供了有力工具。
Description
技术领域
本发明涉及基因工程领域，具体涉及一种用于敲除人ALDH2基因的gRNA序列、ALDH2基因缺失细胞株的构建方法及其应用。
背景技术
ALDH2是乙醛脱氢酶非常重要的一个亚型，位于人体第12号染色体上(12q24.2)，主要作用是将乙醛氧化成乙酸，其参与酒精的次级代谢产物乙醛在体内的代谢和解毒过程，促进酒精在肝脏中代谢，被认为与亚洲人的酒精代谢及酒精依赖性有密切的关联。
慢病毒系统是目前介导目的序列进入靶细胞的方法之一，是以HIV为基础发展起来的一种方法，它克服了部分病毒系统只能感染分裂期细胞的缺陷，慢病毒系统既可以感染分裂期又可以感染非分裂期细胞，并能将目的基因整合进宿主细胞基因组中，发挥持久而稳定的干扰效应。
有限稀释法挑选单克隆细胞的原理是利用梯度稀释，当稀释梯度足够高时，可以获得的单一细胞，培养增殖之后能获得背景单一的细胞株。有限稀释法的操作简单，容易得到单克隆细胞株。
CRISPR/Cas9系统是一种后天免疫防御系统，用以保护细菌或古细菌免受外来质粒或噬菌体的侵入，这类细菌或古细菌基因组的CRISPR序列能表达与入侵者基因组序列相识别的RNA，在CRISPR相关酶(CAS9)的作用下切割外源基因组DNA，达到抵制入侵的目的，经过人为改造后，CRISPR/Cas9系统可以实现在真核细胞中高度灵活且特异的基因组编辑，是目前基因组编辑领域最受欢迎的新一代基因组编辑技术，目前该技术已被用于构建各类基因敲除细胞系和基因敲除动物模型。
现有实验技术中，与CRISPR/Cas9系统最为相似的是siRNA靶向的基因沉默技术siRNA是指生物在进化过程中高度保守的双链RNA诱发的基因沉默现象，其影响生物体一系列过程和功能，但在药物等的诱导作用下，被沉默的基因会出现表达回复的现象。与siRNA靶向ALDH2mRNA抑制基因表达相比，CRISPR/Cas9在ALDH2基因组水平进行基因编辑，可以完全沉默基因的表达，构建真正的ALDH2基因缺陷型细胞株。
目前，有必要提供一种不仅能实现沉默彻底、且能长期稳定体外培养的ALDH2基因缺陷型人肝癌细胞株。
发明内容
为解决上述问题，本发明提供了用于敲除人ALDH2基因的sgRNA序列、ALDH2基因缺失细胞株的构建方法及其应用。
第一方面，本发明提供了一种用于敲除人ALDH2基因的sgRNA序列，所述sgRNA的靶DNA序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示序列中的至少一条。
第二方面，本发明提供了一种敲除人胚胎肾脏细胞ALDH2基因的方法，为利用CRISPR/Cas系统在人肝癌细胞中对ALDH2基因进行改造，具体包括如下步骤：
(1)人工合成如第一方面所述靶DNA序列及其互补链；
(2)将所合成的核酸片段插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
(3)将sgRNA重组质粒转染人肝癌细胞，即得敲除ALDH2基因的人胚胎肾脏细胞。
优选地，所述步骤(2)具体包括：将所合成的SEQ ID NO:1、SEQ ID NO:2及SEQ IDNO:3所示序列的核酸对分别插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
所述步骤(3)具体包括：将步骤(2)所得的两种sgRNA重组质粒共转染人肝癌细胞，即得敲除ALDH2基因的人肝癌细胞。
第三方面，本发明提供了两种ALDH2基因缺失细胞株的构建方法，分别为采用慢病毒转染法、有限稀释法对第二方面所得的敲除ALDH2基因的人肝癌细胞进行传代筛选，获得稳定敲除ALDH2基因的人肝癌细胞。
第四方面，本发明提供了两种ALDH2基因缺失细胞株，为如采用第三方面所述的ALDH2基因缺失细胞株的构建方法所制得。
第五方面，本发明提供了一种如第一方面所述的用于敲除人ALDH2基因的sgRNA序列在敲除ALDH2基因中的应用。
第六方面，一种用于在人基因组中进行ALDH2基因定点敲入试剂盒，包括如下(1)-(3)中的任一种：
(1)如第一方面所述的用于敲除人ALDH2基因的sgRNA序列；
(2)如第二方面所述的sgRNA重组质粒；
(3)如第四方面所述的ALDH2基因缺失细胞株。
本发明提供了的技术方案具有如下有益效果：
本发明提供的技术方案利用CRISPR/Cas9技术对ALDH2基因敲除，实现了在基因组水平对基因的沉默作用，有效改进了siRNA在mRNA或蛋白水平的沉默不彻底或者无法沉默基因表达的缺点。
附图说明
图1为本发明实施例提供的实验设计流程图；
图2为本发明实施例提供的SgRNA重组质粒构建模式图；
图3为本发明实施例提供的单克隆细胞有限稀释法筛选HepG2敲除细胞株测序结果；
图4为本发明实施例提供的SURVEYOR核酸酶消化后PCR片段结果；
图5为本发明实施例提供的ALDH2基因敲除的HepG2稳定株T-A克隆测序结果；
图6为本发明实施例提供的ALDH2基因敲除的HepG2细胞株mRNA表达量检测结果；
图7为本发明实施例提供的ALDH2基因敲除的HepG2细胞株Protein表达量检测结果。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
本发明实施例中无特别说明外，所用试剂及耗材均为市售商品。
本发明技术方案结合图1所示的实验设计流程图，通过如下实施例实现：
(1)sgRNA设计：
分别针对ALDH2基因的第二、第四和第五外显子(Exon2,Exon4,Exon5)设计sgRNA序列。
设计、合成的三组分别针对ALDH2基因第二、第四和第五外显子的sgRNA序列具体分组与命名见表1：
表1 ALDH2sgRNA oligo序列
(Tab.1 The sequences of ALDH2sgRNA oligo)
分别在sgRNA两端加酶切位点，在每条sgRNA序列的正义链的5’端添加CACC，反义链的5’端添加AAAC，从而形成与PX461质粒经Fast Digest Bbs I酶切后互补的粘性末端。如果正义链的5’端第一个碱基不是G，则在5’端CACC后面增加一个G，相应的反义链3’端再增加一个C。设计完成后的sgRNA送上海杰瑞公司进行引物合成。
本发明SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示序列分别对应表格1中SEQID NO:4、SEQ ID NO:6、SEQ ID NO:8画横线部分，具体地，SEQ ID NO:1、SEQ ID NO:2、SEQID NO:3所示序列分别为CCAGTGGACGGATTGACGGT、CATAGGGCTTGCCATTGTCC、CTGAAGAAGTCTCCGTCAAT。
(2)重组质粒的构建与鉴定，构建流程模式如图2所示
①PX458和Lenticrispr V2都是含有U6启动子的sgRNA骨架表达载体，表达具有Cas9D10A切口酶突变的Cas9n，PX458质粒带有GFP绿色荧光蛋白基因和氨苄青霉素抗性，Lenticrispr V2质粒带有氨苄青霉素抗性。用Fast Digest Bbs I对PX458和LenticrisprV2进行酶切，DNA凝胶电泳后回收线性化的载体。
②用T4PNK分别对表1中的三组sgRNA oligo序列进行磷酸化和退火；用T4ligase将线性的线性的PX458和Lenticrispr V2质粒载体分别与退火后的三组sgRNA双链序列室温连接1h。连接产物转化感受态细菌Trans 109，冰浴30min,42℃45s，冰上2min。在氨苄抗性的LB平板上筛选克隆。挑取阳性克隆摇菌，送测序。测序引物为U6启动子的正向引物序列，5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO:15)。测序正确的克隆提取重组质粒。
③所得重组质粒共有六组，针对PX458质粒所得重组质粒命名为PX458-Exon2、PX458-Exon4和PX458-Exon5，针对Lenticrispr V2质粒所得重组质粒命名为LenticrisprV2-Exon2、Lenticrispr V2-Exon4和Lenticrispr V2-Exon5。
(3)慢病毒系统病毒液构建HepG2稳定细胞株
①病毒的产生
293FT细胞培养条件：DMEM培养基(含10％胎牛血清)、5％CO2、37℃恒温培养。
分别在10cm细胞培养皿中接种293FT细胞，细胞接种数为2×106，按照核心质粒：包装质粒：包膜质粒＝4:3:2的比例进行转染，sgRNA组核心质粒分别为Lenticrispr V2-Exon2、Lenticrispr V2-Exon4和Lenticrispr V2-Exon5,以Lenticrispr v2作为对照组。在转染后24h，48h和72h收及病毒液，利用倒置荧光显微镜观察荧光细胞百分比,确定转染效率。将收集的三次病毒液混匀，使用Milipore 100KD浓缩柱对收集的病毒液进行浓缩。利用293FT细胞进行病毒液滴度测定。
②细胞感染和HepG2稳定株细胞筛选。
HepG2细胞培养条件是DMEM培养基(含10％胎牛血清)、5％CO2、37℃恒温培养。
6孔板内提前一天种好待感染的目的细胞株，细胞感染是细胞汇集率达到40-50％为宜。感染前吸去培养基，用2ml新鲜培养基稀释120μl病毒浓缩液，加入3.5g聚凝胺，于感染后48～96h在倒置荧光显微镜下观察感染效率。感染后96h使用终浓度为2μg/ml的嘌呤霉素进行筛选，筛选时间为7天。经嘌呤霉素筛选后用终浓度为0.5μg/ml的嘌呤霉素维持HepG2稳定细胞株。
(4)单克隆细胞有限稀释法筛选HepG2敲除细胞株
①细胞转染：转染前24h，将HepG2细胞以5×105/孔接种至6孔板中培养，转染时细胞融合度达到60％-70％。使用Viafect转染试剂分别将PX458-Exon2、PX458-Exon4和PX458-Exon5同时转染一孔HepG2细胞，等量的PX458质粒作为阴性对照，6孔板质粒转染量一般为2ug/孔,质粒预转染试剂之比为1:2-2.5。24h后观察转染效率。利用倒置荧光显微镜观察荧光细胞百分比，以确定转染效率。
②单克隆细胞有限稀释法筛选单克隆细胞：用胰蛋白酶消化细胞并计数，采用有限稀释法稀释至100ul培养基0.5个细胞，按每孔100ul细胞稀释液加至96孔板中。接种后第5至7天用显微镜观察细胞生长状况并初步筛选出单克隆细胞，待细胞长满96孔板底时，再用胰蛋白酶消化细胞并转移至24孔板中。待细胞长满24孔板底时，一部分细胞用于传代留种，一部分细胞提取其基因组DNA，经PCR扩增后测序，测序结果与原基因组进行比对，检测靶向敲除ALDH2基因是否成功。
测序结果，比对野生型，HepG2敲除细胞株发生了一段4bp碱基的缺失，如图3所示。
(5)提取细胞基因组DNA
将所构建的细胞株进行消化，用GeneJETTM Genomic DNA Purification Kit提取基因组DNA。
①将细胞收集于离心管，每管5X106个细胞，用移液器缓慢吹打，250g离心5min，弃上清，加PBS重悬细胞，再次重复离心，已去除细胞中残留培养基。
②用200ul PBS重悬细胞，每管加入200ul裂解液和20ul蛋白酶K,充分震荡、混合均匀。
③56℃摇床孵育10min，期间每3-4分钟震荡混匀一次，以保证细胞裂解充分。
④加入20ul RNAase A，震荡混匀，室温孵育10min。
⑤加入400ul 50％ethanol，用枪震荡混匀或者震荡混匀。
⑥将上述MiX加入试剂盒提供的Column柱中，6000g离心，1min，将DNA收集柱转移至新的2ml收集管中。
⑦加入500ul wash bufferⅠ，8000g离心1min，弃废液。再加入500ul wash bufferⅡ,12000g,离心3min。
⑧加入200ul Elution Buffer至收集柱滤膜中央，室温孵育2min，8000g离心，1min，即可得所需DNA样品。
(6)PCR反应条件和SURVEYOR分析检测
①根据NCBI提供的ALDH2基因mRNA序列号设计QRT-PCR引物；通过Primer 3.0设计ALDH2基因的SURVEYOR引物和PCR引物，引物由上海捷瑞生物公司合成。
表2 SURVEYOR PCR反应引物序列
Tab.2 PCR primer
②用Phusion超保真DNA聚合酶进行PCR扩增，参照说明书50ul体系，基因组DNA100ng，50ul反应体系如下表所示。
| 组分 | 数量(uL) |
| H2O | to 50 |
| Phusion HF buffer,5X | 10 |
| dNTPs,2.5mM | 4 |
| Phusion polymerase | 0.5 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| template DNA | 100ng |
| Total | 50 |
程序如下：
取反应后PCR产物5ul进行琼脂糖凝胶电泳检测其特异性。
SURVEYOR分析步骤如下：
1)用QIAquick PCR purification Kit试剂盒进行PCR产物纯化，将回收产物稀释至40ng/ul，按照SURVEYOR分析试剂盒说明书步骤进行检测.
2)DNA杂化双链形成(退火反应)体系：
| 组分 | 数量(μl) |
| Taq PCR buffer,10× | 2 |
| Normalized PCR product,20ngμl<sup>-1</sup> | 18 |
| Total volume | 20 |
反应条件：
| 循环次数 | 条件 |
| 1 | 95℃,10min |
| 2 | 95-85℃,-2℃s<sup>-1</sup> |
| 3 | 85℃,1min |
| 4 | 85-75℃,-0.3℃s<sup>-1</sup> |
| 5 | 75℃,1min |
| 6 | 75-65℃,-0.3℃s<sup>-1</sup> |
| 7 | 65℃,1min |
| 8 | 65-55℃,-0.3℃s<sup>-1</sup> |
| 9 | 55℃,1min |
| 10 | 55-45℃,-0.3℃s<sup>-1</sup> |
| 11 | 45℃,1min |
| 12 | 45-35℃,-0.3℃s<sup>-1</sup> |
| 13 | 35℃,1min |
| 14 | 35-25℃,-0.3℃s<sup>-1</sup> |
| 15 | 25℃,1min |
| 16 | 25-4℃,-0.3℃s<sup>-1</sup> |
| 17 | 4℃,hold |
3)SURVEYOR核酸酶消化(在冰上操作)：
反应体系：
| 组分 | 用量(μl) | 终浓度 |
| Annealed heteroduplex | 20 | |
| MgCl<sub>2</sub>stock solution supplied with kit,0.15M | 2.5 | 15mM |
| ddH<sub>2</sub>O | 0.5 | |
| SURVEYOR nuclease S | 1 | 1× |
| SURVEYOR enhancer S | 1 | 1× |
| Total | 25 |
反应条件：充分震荡、混匀上述mixture,42℃30min.
4)取10ul样品，用2％琼脂糖凝胶进行分析。用凝胶定量软件计算切割效率，公式为
fcut＝(b+c)/(a+b+c)，其中Indel为缺失比率，fcut为切割比率，a为未被切割条带的灰度值，b和c表示切割产生的新条带的灰度值。
选择Indel(％)最高的那一组转染的293FT细胞做下一步的接种和筛选。
SURVEYOR核酸酶消化部分结果如图4所示，实验结果表明
1)有限稀释法HepG2敲除株
针对Exon2、Exon4、Exon5设计的sgRNA构建的PX458-Exon2、PX458-Exon4和PX458-Exon5质粒组均有效，SURVEYOR结果为阳性。
2)慢病毒转染法HepG2敲除株
针对Exon2、Exon5设计的sgRNA构建的PX458-Exon2和PX458-Exon5质粒组均有效，SURVEYOR结果为阳性。
(7)ALDH2基因敲除的HepG2细胞株DNA测序分析
①提取细胞基因组DNA，使用Phusion高保真酶进行PCR，并对PCR产物进行纯化，使用Taq DNA聚合酶对上述PCR产物加A尾，反应总体系为50μl，反应具体所需各组分使用量和反应条件如下：
②连接T载体：将上述加A尾的PCR产物与T载体进行连接，同时使用T载体试剂盒自带的对照设置阴性对照，反应所需的各组分量如下所示：
对照组如下：
上述反应体系中每管加入SolutionⅠ(内含连接反应所需的酶)5μl，于16℃连接30min，整个反应在PCR仪上进行。
③连接产物转化感受态细菌Trans 109，冰浴30min,42℃45s，冰上2min。在氨苄抗性的LB平板上筛选克隆。挑取阳性克隆摇菌，送北京六合华大基因公司测序。
结果如图5所示，HepG2稳定株在靶点位置造成了的缺失突变，实现ALDH2基因敲除。
(8)ALDH2基因敲除的HepG2细胞株mRNA和蛋白表达量检测
①使用Trizol法提取细胞总RNA，使用TOYOBO公司逆转录试剂盒进行逆转录，生成cDNA，按照TOYOBO公司SYBR mix试剂说明书给出的体系进行qRT-PCR。
结果如图6所示，成功构建ALDH2基因敲除的HepG2细胞株mRNA表达量明显低于对照组。
②使用M-PER细胞裂解液提取HepG2ALDH2基因敲除细胞株总蛋白，使用BCA蛋白定量试剂盒进行定量，进行Western Blot。
结果如图7所示，成功构建ALDH2基因敲除的HepG2细胞株蛋白表达量明显低于对照组。
相对于传统的基因敲除方法不仅流程繁琐，对技术要求高，而且费用昂贵，成功率相对较低。本发明采用的CRISPR-Cas9技术是第四代基因编辑方法，其易于操作，效率更高，费用低廉。本发明利用CRISPR-Cas9技术构建的ALDH2基因敲除细胞模型为ALDH2代谢相关的研究提供了有效平台。具体有益效果如下：
(1)利用CRISPR/Cas9技术对ALDH2基因敲除，实现了在基因组水平对基因的沉默作用，有效改进了siRNA在mRNA或蛋白水平的沉默不彻底或者无法沉默基因表达的缺点。
(2)ALDH2参与机体重要的代谢功能，ALDH2基因敲出细胞株的建立为外源性化学物或者外源性毒物在体内的代谢研究提供了有效的平台。
(3)ALDH2基因敲除细胞株对于慢性疾病(如酒精性肝脏疾病及糖尿病)及肿瘤相关疾病的研究提供了有力工具。
(4)ALDH2基因敲除细胞株可用于ALDH2代谢相关外源性化学物毒性、致癌性的研究以及药物之间的交互作用研究。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中山大学
<120> 用于敲除人ALDH2基因的sgRNA、ALDH2基因缺失细胞株的构建方法及应用
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Claims (6)
1.一种用于敲除人ALDH2基因的sgRNA序列，其特征在于，所述sgRNA的靶DNA序列为SEQID NO:1、SEQ ID NO:2、SEQ ID NO:3所示序列中的至少一条。
2.一种敲除人肝癌细胞ALDH2基因的方法，其特征在于，为利用CRISPR/Cas系统在人肝癌细胞中对ALDH2基因进行改造，具体包括如下步骤：
(1)人工合成如权利要求1所述的靶DNA序列及其互补链；
(2)将所合成的核酸片段插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
(3)将sgRNA重组质粒转染人肝癌细胞，即得敲除ALDH2基因的人肝癌细胞。
3.一种ALDH2基因缺失细胞株的构建方法，其特征在于，采用慢病毒转染法或有限稀释法对如权利要求2所得的敲除ALDH2基因的人肝癌细胞进行传代筛选，获得稳定敲除ALDH2的人肝癌细胞。
4.一种ALDH2基因缺失细胞株，其特征在于，为采用如权利要求3所述的ALDH2基因缺失细胞株的构建方法所制得。
5.一种如权利要求1所述的用于敲除人ALDH2基因的sgRNA序列在敲除ALDH2基因中的应用。
6.一种用于在人基因组中进行ALDH2基因定点敲除试剂盒，其特征在于，包括如下(1)-(3)中的任一种：
(1)如权利要求1所述的用于敲除人ALDH2基因的sgRNA序列；
(2)如权利要求2所述的sgRNA重组质粒；
(3)如权利要求4所述的ALDH2基因缺失细胞株。
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