CN115521899A - 一种高效的日本青鱂体外培养细胞CRISPR/Cas9基因组编辑方法及其应用 - Google Patents
一种高效的日本青鱂体外培养细胞CRISPR/Cas9基因组编辑方法及其应用 Download PDFInfo
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Abstract
本发明涉及日本青鱂体外培养细胞的一种高效基因组编辑方法及其应用。本发明基于CRISPR/Cas9基因组编辑技术,用日本青鱂内源U6启动子驱动的gRNA、sCMV启动子驱动的基于斑马鱼密码子优化的Cas9(zCas9)以及SV40启动子驱动的G418抗性筛选基因NeoR,构建NeoR‑gRNA‑zCas9表达载体,通过质粒转染日本青鱂体外培养细胞及G418筛选,以实现体外培养细胞的基因组高效编辑和筛选。相较于目前已有鱼类体外培养细胞的基因组编辑技术体系,成本低、操作简单、效率高。该发明为日本青鱂体外培养细胞的基因组编辑提供了一种强有力的研究工具,对其它鱼类体外培养细胞的基因组编辑具有借鉴意义。
Description
技术领域
本发明属于分子生物学领域,涉及日本青鱂体外培养细胞的一种高效基因组编辑方法体系。
背景技术
体外培养细胞是病毒学、环境毒理学、遗传育种、资源保护、分子机制等研究的有用模型。与活体相比,体外培养细胞不仅可以节省成本和时间,而且可以最大限度地减少遗传异质性和其他影响因素的影响,如神经内分泌因素、与其他细胞的相互作用、伦理问题等。近年来,CRISPR/Cas9(Clustered regularly interspaced short palindromicrepeats- CRISPR-associated protein 9)已成功应用于哺乳动物体外培养细胞的基因组编辑,如人类、小鼠、大鼠,并且已经建立了多种基因敲除和敲入哺乳动物细胞系,这些研究对基因功能分析、体内细胞追踪、高通量识别影响细胞生物学过程的潜在因素等均具有重要意义。在鱼类, CRISPR/Cas9系统成功应用于斑马鱼、罗非鱼、草鱼、南方鲶鱼和草鱼等多种鱼类的在体基因组编辑,然而,其在鱼类体外培养细胞的基因组编辑一直以来未能获得满意结果,其应用受到了极大限制。2016年Carola等报道,首先通过获得Cas9稳定表达的奇努克鲑鱼 (Oncorhynchus tshawytscha)体外培养细胞,然后瞬时转染体外合成的gRNA,虽然也有获得基因组编辑的阳性细胞,但其效率低下。2016年Liu等报道,通过电穿孔法将预先形成的 gRNA-Cas9核糖核蛋白(RNP)转染到日本青鱂培养的细胞中,获得了基因编辑阳性细胞。该方法需要购买RNP等价格昂贵试剂及特殊设备电穿孔仪,并且需要对电穿孔等相关参数进行优化,实验成本高,操作复杂,技术要求高。一种高效、经济、简便的日本青鱂体外培养细胞的基因组编辑方法亟待开发。
发明内容
本发明的主要目的之一在于建立一种适用于日本青鱂体外培养细胞中的CRISPR/Cas9 基因编辑技术体系;目的之二在于提供一种上述质粒的构建方法;目的之三在于提供一种基于上述体系进行日本青鱂体外培养细胞中的基因编辑的方法。
为达到上述目的,本发明提供如下技术方案:
一种基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,包括G418筛选基因表达模块、gRNA表达模块和Cas9表达模块,所述G418筛选基因表达模块、gRNA表达模块和Cas9表达模块构建到同一个表达载体中,构建pzCas9-OlU6-gRNA质粒。
所述G418筛选基因表达模块为新霉素抗性基因(NeoR)表达框,由SV40启动子驱动NeoR基因,由SV40 poly(A)终止子终止。
所述SV40启动子的DNA序列如SEQ ID NO.1所示;
所述NeoR基因的DNA序列如SEQ ID NO.2所示;
所述SV40 poly(A)终止子的DNA序列如SEQ ID NO.3所示。
所述gRNA表达模块为gRNA表达框,带有gRNA scaffold序列,由日本青鳉内源U6核小RNA(OlU6)启动子启动。
所述OlU6启动子的DNA序列如SEQ ID NO.4所示;
所述gRNA scaffold的DNA序列如SEQ ID NO.5所示。
所述zCas9表达模块为基于斑马鱼密码子优化的Cas9基因(zCas9)表达框,由sCMV启动子驱动zCas9,带有非洲爪蟾β-珠蛋白3’UTR序列,由bGH poly(A)信号终止。
所述sCMV启动子的DNA序列如SEQ ID NO.6所示;
所述zCas9基因的DNA序列如SEQ ID NO.7所示;
所述非洲爪蟾β-珠蛋白3’UTR的DNA序列如SEQ ID NO.8所示;
所述bGH poly(A)终止信号的DNA序列如SEQ ID NO.9所示。
基于一个总的技术路线构思,本发明还提供了上述CRISPR/Cas9编辑系统的使用方法,具体而言,包括以下步骤:
(1)构建所述含有目标基因gRNA序列的质粒,pzCas9-OlU6-gRNA。
(2)将所述质粒转染至青鳉体外培养细胞,得到转染细胞。
(3)将转染的细胞培养筛选得到基因编辑的细胞,并进行检测。
在上述CRISPR/Cas9编辑系统的使用方法中,步骤(1)具体为:
(a)设计能够特异性靶向日本青鳉内源基因的gRNA。
(b)依据gRNA序列设计包含gRNA序列的同源重组用引物,gRNA-F和gRNA-R。
(c)依据载体序列设计连接载体用的同源重组引物,所述Mlu-F引物的DNA序列如SEQ ID NO.10所示,所述Sal-R引物的DNA序列如SEQ ID NO.11所示。
(d)使用引物Mlu-F和gRNA-R,以pzCas9-OlU6-gRNA质粒为模板,进行PCR扩增并回收;使用引物gRNA-F和Sal-R,以pzCas9-OlU6-gRNA质粒为模板,进行PCR扩增并回收。
(e)将通过限制酶MluⅠ和SalⅠ双酶切的pzCas9-OlU6-gRNA质粒片段与上述步骤回收的2个片段进行同源重组连接并转化大肠杆菌感受态DH5α。
(f)挑取转化得到的细菌进行单克隆细菌扩增培养,分离得到质粒。
在上述CRISPR/Cas9编辑系统的使用方法中,步骤(2)具体为:
使用pzCas9-OlU6-gRNA质粒转染日本青鳉体外培养细胞。
在上述CRISPR/Cas9编辑系统的使用方法中,步骤(3)具体为:
转染后的SG3细胞培养一段时间后在培养皿中加入G418使其终浓度为1000ug/mL,每 3天更换一次含有1000ug/mL G418的培养基,7天后细胞基本稳定,得到基因编辑后的细胞系。
同时,本发明提供了一种基于上述CRISPR/Cas9编辑系统编辑日本青鳉patched 1基因的使用实例,具体而言,步骤如下:
(a)设计能够特异性靶向日本青鳉内源基因patched 1的gRNA,patched1-gRNA1(SEQ ID NO.12)和patched1-gRNA2(SEQ ID NO.13)。
(b)依据gRNA序列设计包含gRNA序列的同源重组用引物,patched1-gRNA1-F(SEQID NO.14)和patched1-gRNA1-R(SEQ ID NO.15)、patched1-gRNA2-F(SEQ ID NO.16)和patched1-gRNA2-R(SEQ ID NO.17)。
(c)使用引物Mlu-F和patched1-gRNA1-R,以pzCas9-OlU6-gRNA质粒为模板,进行PCR扩增并回收片段1;使用引物patched1-gRNA1-F和Sal-R,以pzCas9-OlU6-gRNA质粒为模板,进行PCR扩增并回收片段2。
使用引物Mlu-F和patched1-gRNA2-R,以pzCas9-OlU6-gRNA质粒为模板,进行 PCR扩增并回收片段3;使用引物patched1-gRNA2-F和Sal-R,以pzCas9-OlU6-gRNA质粒为模板,进行PCR扩增并回收片段4。
(e)将通过限制酶MluⅠ和SalⅠ双酶切的pzCas9-OlU6-gRNA质粒片段与上述步骤回收的片段1和片段2进行同源重组连接并转化大肠杆菌感受态DH5α。
将通过限制酶MluⅠ和SalⅠ双酶切的pzCas9-OlU6-gRNA质粒片段与上述步骤回收的片段 3和片段4进行同源重组连接并转化大肠杆菌感受态DH5α。
(f)挑取转化得到的细菌进行单克隆细菌扩增培养,分离得到质粒pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2。
(g)使用上一步得到的质粒转染日本青鳉体外培养细胞SG3,转染后两天加入1000μg/mL的G418,每3天更换新的含有1000μg/mL的G418的培养基。
(g)G418筛选7天后,收取细胞提取基因组并用检测。
本发明构建出的日本青鳉体外培养细胞CRISPR/Cas9基因编辑载体具有以下优势:
(1)本发明提供了一种基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,通过PCR扩增以及无缝克隆即可获得含有目的基因靶点序列的载体质粒,获得的质粒只需要转染细胞即可完成特异靶点的基因组编辑工作,相比于现有的鱼类体外培养细胞中的基因编辑方法,克服了直接使用gRNA转染的不稳定性,克服了构建稳转Cas9 细胞系的繁琐工作,同时也克服了如RNP转染中高成本、技术要求高等问题。
(2)本发明提供的基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体中gRNA的表达由青鳉内源U6启动子启动,相较于人源U6以及斑马鱼U6启动子,该内源U6启动子能够在日本青鳉体外培养细胞中高效启动gRNA的表达,极大的提高了基因组编辑的效率。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为本发明所述CRISPR/Cas9基因编辑载体质粒图谱结构图。
图2为本发明所述载体构建技术流程图。
图3为本发明所涉及靶点序列及其在基因组上位置信息示意图。
图4为对本发明所涉及靶点进行基因编辑后的异源双链泳动分析的PAGE图。
具体实施方式
实施例1:构建用于敲除日本青鳉patched1基因的pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2质粒。
结合图1和图2所示,基于CRISPR/Cas9系统编辑日本青鳉体外培养细胞基因的载体的构建方法,具体步骤如下:
(1)利用gRNA在线设计网站(http://crispr.dbcls.jp/)在日本青鳉patched 1基因第1个和第2个外显子上设计特异性的gRNA,如图3所示。
patched1-gRNA1:GCGTTATCCTCGGCCGATCA(SEQ ID NO.12);
patched1-gRNA2:ACGTTGGCTCCCTCTTCGCG(SEQ ID NO.13)。
根据设计好的gRNA序列设计含有gRNA的同源重组用引物。
patched1-gRNA1-F:GCGTTATCCTCGGCCGATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.14);
patched1-gRNA1-R:TGATCGGCCGAGGATAACGCCGATGAGCCAAAGTCTCTGAG(SEQ IDNO.15);
patched1-gRNA2-F:ACGTTGGCTCCCTCTTCGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.16);
patched1-gRNA2-R:CGCGAAGAGGGAGCCAACGTCGATGAGCCAAAGTCTCTGAG(SEQ IDNO.17)。
(2)使用Mlu-F和patched1-gRNA1-R引物以pzCas9-OlU6-gRNA质粒为模板进行PCR扩增反应,获得PCR产物1;
使用patched1-gRNA1-F和Sal-R引物以pzCas9-OlU6-gRNA质粒为模板进行PCR扩增反应,获得PCR产物2;
使用Mlu-F和patched1-gRNA2-R引物以pzCas9-OlU6-gRNA质粒为模板进行PCR扩增反应,获得PCR产物3;
使用patched1-gRNA2-F和Sal-R引物以pzCas9-OlU6-gRNA质粒为模板进行PCR扩增反应,获得PCR产物4;
所用PCR体系为:PrimeSTAR Max Premix(2×)25μL,正反向引物各1.5μL,模板1μL(50ng),加入21μL的ddH2O补足至总体积50μL。
所用PCR反应程序为:①98℃预变性30s;②32循环(98℃10s,55℃5s,72℃10s);③72℃终延伸5min;④4℃保存。
琼脂糖凝胶电泳回收PCR产物1、PCR产物2、PCR产物3和PCR产物4,分别得到片段1、片段2、片段3和片段4。
(3)使用Takara公司的SalⅠ酶和MluⅠ酶对pzCas9-OlU6-gRNA质粒进行双酶切并进行琼脂糖凝胶电泳回收,获得长约9kb的线性化载体片段。
双酶切体系为:质粒3μg,SalⅠ酶和MluⅠ酶各10U,10×H buffer 5μL,ddH2O补齐至总体积50μL。
双酶切条件为37℃水浴酶切3h。
(4)将线性化的载体片段与片段1和片段2进行酶促组装得到 pzCas9-OlU6-patched1gRNA1;将线性化的载体片段与片段3和片段4进行酶促组装得到pzCas9-OlU6-patched1gRNA2。
酶促组装体系:全式金2×Basic Assembly Mix 5μl,线性化载体约100ng,片段1或3 约35ng,片段2或4约8ng,补足ddH2O至总体积10μL。
反应条件:50℃,20min;4℃保存。
(5)将酶促组装产物pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2转化大肠杆菌感受态细胞。所得转化产物涂布到含有氨苄青霉素抗性的LB平板上过夜培养。第二天挑取单菌落,使用引物Mlu-F和Sal-R进行菌落PCR检测,筛选条带大小为1000bp左右的单菌落并送测序。
测序结果显示pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2质粒含有靶点的完整gRNA表达框。
(6)挑取单克隆细菌扩增培养,并用TIANGEN无内毒素质粒小提中量试剂盒提取质粒。
实施例2:日本青鱂体外培养细胞中patched 1基因编辑方法
(1)转染前一天将生长状态良好的日本青鳉SG3细胞铺板于24孔细胞培养板中,铺板密度以第二天转染时细胞密度生长到80%左右为宜。
(2)第二天使用Mirus公司的动态传递系统分别转染 pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2质粒到SG3细胞中。转染严格按照试剂配套的说明书进行。
(3)转染后48小时在培养皿中添加G418并使其终浓度为1000ng/mL,每3天使用含1000ng/mL G418的新鲜培养基替换旧的培养基,G148筛选7天后收取细胞并提取全基因组。
(4)使用PAGE电泳进行异源双链泳动分析来检测突变。设计针对不同靶点的检测引物,使用检测引物同时对正常细胞提取的基因组和转染质粒后细胞提取的基因组进行PCR扩增, PCR产物进行PAGE电泳,电泳结果显示,转染pzCas9-OlU6-patched1gRNA1和pzCas9-OlU6-patched1gRNA2质粒的细胞基因组的扩增产物出现了大小不一的条带如图4,表明转染质粒后出现了基因突变,即成功对patched 1基因进行了基因编辑。
所用PCR体系为:Vazyme 2×Taq Master Mix 25μL,正反向引物各1.5μL,模板1μL(200 ng),加入21μL的ddH2O补足至总体积50μL。
所用PCR反应程序为:①95℃预变性3min;②35循环(95℃30s,55℃30s,72℃60s);③72℃终延伸5min;④4℃保存。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其做出适当改变,而不偏离本发明权利要求书所限定的范围。
SEQUENCE LISTING
<110> 西南大学
<120> 日本青鱂体外培养细胞的一种高效、简便的CRISPR/Cas9基因组编辑方法及
其应用
<130> 2022
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 358
<212> DNA
<213> 人工序列
<400> 1
ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 60
ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 120
aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 180
aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 240
ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 300
ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaa 358
<210> 2
<211> 795
<212> DNA
<213> 人工序列
<400> 2
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 3
<211> 135
<212> DNA
<213> 人工序列
<400> 3
aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120
tatcatgtct ggatc 135
<210> 4
<211> 409
<212> DNA
<213> 人工序列
<400> 4
gtaaccacca acacatgggg gttggttgga gctgctgtgg ggattggtaa aggtcctcct 60
ggaggggtgt tgacttgacc ttttgttctg gcgcttccgg gaaaccagtc gcactcgtct 120
gacgtgacaa aacccccctt cttttcccag aatccttcca agaagtttct cacaaagtca 180
gaaaaaatgt aattttaatc tgatcagaaa catttaagtc aaactttaaa tggagatggt 240
gaacctgaga cgatcctgtg tcatgggaga aacagatgat cctttttggt gttttgtcat 300
cattgtggtt ccctttaaag tccactgcgt tccctcagat cacacctcat gcttgagagg 360
agtagcagag gggagggttt aaaaagcact cagagacttt ggctcatcg 409
<210> 5
<211> 76
<212> DNA
<213> 人工序列
<400> 5
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgc 76
<210> 6
<211> 978
<212> DNA
<213> 人工序列
<400> 6
gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt 60
gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca 120
gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt 180
gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg 240
tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat 300
gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca 360
ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt 420
gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac 480
cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac 540
gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta 600
aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa 660
tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg 720
taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac 780
tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg 840
gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa 900
atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc 960
aatgctcgtt tagggaac 978
<210> 7
<211> 4101
<212> DNA
<213> 人工序列
<400> 7
atggataaga agtatagcat cggcctggat attggaacta actccgtggg ttgggcagtg 60
attacagacg actacaaggt ccctagcaag aaatttaagg tgctgggtaa caccgacagg 120
cacagcatca agaaaaatct gattggagcc ctgctgttcg gttctggaga gactgccgaa 180
gcaacacgcc tgaaaagaac agcaagaagg cgctatacca gaaggaagaa tagaatctgt 240
tacctgcagg agattttctc taacgaaatg gctaaggtgg acgattcatt ctttcatagg 300
ctggaggaaa gtttcctggt cgaggaagat aagaaacacg agcgccatcc tatctttgga 360
aacattgtgg acgaggtcgc ctatcacgaa aaatacccaa ccatctatca tctgcgcaag 420
aaactggctg actctactga taaagccgac ctgagactga tctatctggc tctggcccac 480
atgattaagt tcaggggtca ttttctgatc gagggcgatc tgaaccccga caattccgat 540
gtggacaagc tgttcatcca gctggtccag atttacaatc agctgtttga ggaaaaccct 600
attaatgctt ccagagtgga cgcaaaagct atcctgtcag ccaggctgtc caagtcacgc 660
agactggaga acctgattgc acagctgccc ggagaaaaga ggaacggtct gtttggaaat 720
ctgatcgctc tgagtctggg cctgactcct aacttcaaaa gcaattttga tctggctgag 780
gacgccaaac tgcagctgtc aaaggacaca tatgacgatg acctggataa cctgctggca 840
cagatcggag atcagtacgc tgacctgttc ctggctgcca aaaatctgtc cgacgcaatc 900
ctgctgtcag atattctgag agtgaacagc gagattacaa aagcacctct gagtgccagc 960
atgatcaaga gatatgacga gcaccatcag gatctgaccc tgctgaaggc tctggtcagg 1020
cagcagctgc cagagaagta caaggaaatt ttctttgatc agtccaagaa cggctacgcc 1080
ggttatatcg acggaggcgc atcacaggag gaattctaca agtttatcaa acctattctg 1140
gagaagatgg acggaactga ggaactgctg gtgaaactga atagagagga cctgctgagg 1200
aagcagcgca catttgataa cggttccatc ccacaccaga ttcatctggg agagctgcac 1260
gctatcctga ggcgccagga agacttctac ccctttctga aagataaccg cgagaagatc 1320
gaaaaaattc tgaccttcag aatcccttac tatgtgggtc cactggctcg cggaaacagc 1380
agatttgcct ggatgactcg caaatccgag gaaaccatta ctccttggaa cttcgaggaa 1440
gtggtcgata agggcgcctc tgcacagtcc ttcatcgaga gaatgactaa ttttgacaaa 1500
aacctgccca atgagaaagt gctgcctaag cactccctgc tgtacgagta tttcactgtc 1560
tataacgaac tgacaaaggt gaaatacgtc accgagggca tgagaaagcc agccttcctg 1620
tcaggagagc agaagaaagc aatcgtggat ctgctgttta aaaccaacag gaaagtgact 1680
gtcaagcagc tgaaggagga ctacttcaag aaaattgaat gcttcgattc cgtggagatc 1740
agcggagtcg aagacagatt taacgcaagc ctgggcgctt accacgatct gctgaagatc 1800
attaaggata aagacttcct ggacaacgag gaaaatgagg atatcctgga agacattgtg 1860
ctgacactga ccctgtttga ggacagagga atgatcgagg aaagactgaa aacctatgct 1920
catctgttcg atgacaaggt gatgaaacag ctgaagagaa ggcgctacac tggctggggt 1980
agactgagca ggaagctgat caacggcatt agggataaac agtcaggaaa gacaatcctg 2040
gactttctga aaagtgatgg cttcgccaac cgcaatttta tgcagctgat tcacgatgac 2100
agtctgacct tcaaagagga catccagaag gctcaggtgt ctggacaggg ccactccctg 2160
catgagcaga ttgcaaacct ggctggaagc ccagccatca agaaaggcat tctgcagaca 2220
gtgaaaatcg tcgatgagct ggtgaaagtc atgggccata agcccgaaaa catcgtgatt 2280
gagatggctc gcgaaaatca gacaacccag aagggtcaga agaacagtag agagaggatg 2340
aaaagaatcg aggaaggcat taaggagctg ggtagccaga tcctgaaaga gcacccagtg 2400
gaaaacacac agctgcagaa tgagaagctg tatctgtact atctgcagaa tggaagagat 2460
atgtacgtgg accaggagct ggatattaac aggctgtctg attacgacgt ggatcatatc 2520
gtcccccaga gtttcatcaa agatgacagc attgacaaca aggtgctgac caggtccgac 2580
aaaaacagag gaaaatcaga taatgtccct agtgaggaag tggtcaagaa aatgaagaac 2640
tactggagac agctgctgaa tgccaaactg atcactcaga ggaagtttga taacctgaca 2700
aaagcagagc gcggtggact gtcagaactg gacaaagctg gattcatcaa gaggcagctg 2760
gtggaaacac gccagatcac taaacacgtc gcacagattc tggatagtcg catgaacaca 2820
aagtacgatg agaatgacaa actgatcaga gaagtgaagg tcattaccct gaagagtaaa 2880
ctggtcagcg actttaggaa agatttccag ttttataagg tccgcgagat taacaattat 2940
caccatgccc atgacgcata cctgaacgcc gtggtcggta ccgcactgat caagaaatac 3000
ccaaaactgg agagcgaatt cgtgtacgga gactataagg tgtacgatgt cagaaaaatg 3060
atcgccaagt ccgagcagga aattggaaaa gctactgcca agtatttctt ttactcaaac 3120
atcatgaatt tctttaagac agagatcacc ctggccaatg gagaaatccg caaaaggccc 3180
ctgattgaga caaacggaga gacaggcgaa atcgtgtggg acaaaggcag agattttgca 3240
accgtgagga aggtcctgag catgcctcaa gtgaatatcg tcaagaaaac tgaggtgcag 3300
acaggcggtt tctcaaaaga aagtattctg ccaaaacgca actctgataa gctgatcgct 3360
agaaagaaag actgggaccc taagaagtat ggaggctttg actctcccac tgtggcatac 3420
tccgtcctgg tggtcgctaa ggtggagaag ggcaaaagca agaaactgaa atctgtcaag 3480
gagctgctgg gtatcacaat tatggagaga agctctttcg agaagaaccc aatcgatttt 3540
ctggaggcca aaggttataa ggaagtgaag aaagacctga tcattaaact gcccaagtac 3600
agtctgtttg agctggaaaa cggcaggaaa cgcatgctgg caagcgctgg agagctgcag 3660
aaaggcaatg aactggccct gccttctaag tacgtgaact tcctgtatct ggcaagccac 3720
tacgagaagc tgaaaggatc tccagaggat aacgaacaga aacagctgtt tgtggagcag 3780
cacaagcatt atctggacga gatcattgaa cagattagcg agttctctaa aagagtgatc 3840
ctggccgacg caaatctgga taaggtcctg tctgcttaca acaaacacag agataagccc 3900
atcagggagc aggccgaaaa tatcattcat ctgttcactc tgacaaacct gggcgcacct 3960
gcagctttca agtacttcga cactacaatc gatagaaaga ggtacacctc cactaaggag 4020
gtgctggacg ctacactgat ccatcagagt attaccggcc tgtacgaaac aaggattgac 4080
ctgtctcagc tgggtggcga c 4101
<210> 8
<211> 126
<212> DNA
<213> 人工序列
<400> 8
aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt 60
acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc tcctaataaa aagaaagttt 120
cttcac 126
<210> 9
<211> 225
<212> DNA
<213> 人工序列
<400> 9
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225
<210> 10
<211> 38
<212> DNA
<213> 人工序列
<400> 10
cttgacgagt tcttctgaac gcgtctcgag cctctaga 38
<210> 11
<211> 40
<212> DNA
<213> 人工序列
<400> 11
cgccatattg aattggcggt cgactggcgt aatagccaac 40
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<400> 12
gcgttatcct cggccgatca 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<400> 13
acgttggctc cctcttcgcg 20
<210> 14
<211> 50
<212> DNA
<213> 人工序列
<400> 14
gcgttatcct cggccgatca gttttagagc tagaaatagc aagttaaaat 50
<210> 15
<211> 41
<212> DNA
<213> 人工序列
<400> 15
tgatcggccg aggataacgc cgatgagcca aagtctctga g 41
<210> 16
<211> 50
<212> DNA
<213> 人工序列
<400> 16
acgttggctc cctcttcgcg gttttagagc tagaaatagc aagttaaaat 50
<210> 17
<211> 41
<212> DNA
<213> 人工序列
<400> 17
cgcgaagagg gagccaacgt cgatgagcca aagtctctga g 41
Claims (8)
1.一种基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,包括G418筛选基因表达模块、gRNA表达模块和Cas9表达模块,所述G418筛选基因表达模块、gRNA表达模块和Cas9表达模块构建到同一个表达载体中,构建pzCas9-OlU6-gRNA质粒。
2.根据权利要求1所述的基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,所述G418筛选基因表达模块为新霉素抗性基因(NeoR)表达框,由SV40启动子驱动,由SV40 poly(A)终止子终止;
所述SV40启动子的DNA序列如SEQ ID NO.1所示;
所述NeoR基因的DNA序列如SEQ ID NO.2所示;
所述SV40 poly(A)终止子的DNA序列如SEQ ID NO.3所示。
3.根据权利要求1所述的基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,所述gRNA表达模块为gRNA表达框,带有gRNA scaffold序列,由日本青鳉内源U6核小RNA(OlU6)启动子驱动;
所述OlU6启动子的DNA序列如SEQ ID NO.4所示;
所述gRNA scaffold的DNA序列如SEQ ID NO.5所示。
4.根据权利要求1所述的基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,所述Cas9表达模块为基于斑马鱼密码子优化的Cas9基因(zCas9)表达框,由sCMV启动子驱动,带有非洲爪蟾β-珠蛋白3’UTR序列,由bGH poly(A)信号终止;
所述sCMV启动子的DNA序列如SEQ ID NO.6所示;
所述zCas9基因的DNA序列如SEQ ID NO.7所示;
所述非洲爪蟾β-珠蛋白3’UTR的DNA序列如SEQ ID NO.8所示;
所述bGH poly(A)终止信号的DNA序列如SEQ ID NO.9所示。
5.根据权利要求1所述的基于CRISPR/Cas9系统对日本青鳉体外培养细胞进行基因组编辑的载体构建方法,其特征在于,包括以下步骤:
(1)设计能够特异性靶向日本青鳉内源基因的gRNA;
(2)依据gRNA序列设计包含gRNA序列的同源重组用引物,gRNA-F和gRNA-R;
(3)依据载体序列设计连接载体用的同源重组引物,Mlu-F和Sal-R;
(4)使用引物Mlu-F和gRNA-R,以权利要求1所述质粒为模板,进行PCR扩增并回收;使用引物gRNA-F和Sal-R,以权利要求1所述质粒为模板,进行PCR扩增并回收;
(5)将通过限制酶MluⅠ和SalⅠ双酶切的载体片段与(4)所述回收的2个片段进行同源重组连接并转化大肠杆菌感受态DH5α;
(6)挑取(5)转化得到的细菌进行单克隆细菌扩增培养,分离得到质粒。
6.根据权利要求5所述的基于CRISPR/Cas9系统编辑日本青鳉体外培养细胞的载体的构建方法,其特征在于,所述Mlu-F引物的DNA序列如SEQ ID NO.10所示,所述Sal-R引物的DNA序列如SEQ ID NO.11所示。
7.权利要求1或5所述构建方法获得的基于CRISPR/Cas9体系对日本青鳉体外培养细胞进行基因组编辑的载体。
8.一种在日本青鳉体外培养细胞中进行基因编辑的方法,其特征在于,包括以下步骤:
(1)构建如权利要求1或5所述的质粒;
(2)将所述质粒转染至青鳉体外培养细胞,得到转染细胞;
(3)将转染的细胞培养筛选得到基因组编辑的细胞,并进行检测。
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