CN107893080A - 一种靶向大鼠Inhba基因的sgRNA及其应用 - Google Patents
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Abstract
本发明属于基因工程领域，具体涉及一种靶向识别大鼠Inhba基因的sgRNA及其应用。公开了一种靶向Inhba基因的sgRNA以及利用CRISPR‑Cas9系统编辑大鼠Inhba基因的方法。本发明提供的sgRNA能特异的靶向大鼠Inhba基因，并且能够以25％的效率对Inhba基因进行编辑，为进一步研究大鼠Inhba基因在其卵泡发育中的功能提供了基础。
Description
技术领域
本发明属于基因工程领域，具体涉及一种靶向识别大鼠Inhba基因的sgRNA及其应用。
背景技术
对基因的靶向性编辑是研究基因功能的重要手段。传统的同源重组介导的基因打靶技术在研究基因的功能上发挥了重要作用，但是存在着极低的打靶效率、复杂的载体构建以及冗长的细胞筛选等缺点(Vasquez KM,Marburger K,Intody Z,WilsonJH.Manipulating the mammalian genome by homologous recombination.Proc NatlAcad Sci U S A.2001Jul 17；98(15):8403-10.)，已严重阻碍了目前对高通量及高效靶向基因编辑的需求。近年来以ZFN 和TALEN为代表的人工核酸酶在基因的靶向性编辑中得到了迅速发展，它们通过以一个结合域识别特定DNA位点以及另一个内切酶结构域切割DNA的方式能方便的对基因位点进行编辑，其效率与传统的基因打靶相比得到了大幅度提高(GajT,Gersbach CA,Barbas CF 3rd. ZFN,TALEN,and CRISPR/Cas-based methods forgenome engineering.Trends in Biotechnology. 2013Jul；31(7):397-405.)，然而ZFN和TALEN的构建程序仍十分繁琐，限制了它们的广泛应用。
CRISPR/Cas9系统是细菌和古细菌为抵抗外源病毒入侵所进化的一种适应性免疫防御系统(Horvath P,Barrangou R.CRISPR/Cas,the Immune System ofBacteria andArchaea.Science. 2010Jan 8；327(5962):167-70)。它通过一小段RNA(sgRNA)与DNA识别并借助Cas9核酸酶可以高效精确的对基因进行靶向编辑(Jinek M,Chylinski K,FonfaraI,Hauer M,Doudna JA, Charpentier E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science.2012Aug 17；337(6096):816-21.)。研究表明在高等生物例如人、动植物中也存在CRISPR/Cas9系统，并且已经利用CRISPR/Cas9系统对人、小鼠、植物的基因都进行了有效的编辑(Hsu PD,Scott DA,Weinstein JA,Ran FA,Konermann S,Agarwala V,LiY,Fine EJ,Wu X, ShalemO,CradickTJ,MarraffiniLA,Bao G,ZhangF.DNAtargeting specificity ofRNA-guidedCas9nucleases. NatBiotechnol.2013Sep；31(9):827-32；
WuY,Liang D,WangY,Bai M,TangW,Bao S,Yan Z,Li D,Li J.Correction ofagenetic disease in mouse viauseofCRISPR-Cas9.Cell StemCell.2013Dec 5；13(6):659-62；
Jiang W,Zhou H,Bi H,Fromm M,Yang B,Weeks DP.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghumand rice.NucleicAcids Res.2013Nov；41(20):e188)。此外通过改造Cas9核酸酶，该系统可大幅度的提高靶向特异性，使脱靶编辑降低到无法检测到的水平(Slaymaker IM,Gao L,Zetsche B, Scott DA,Yan WX,Zhang F.Rationally engineered Cas9nucleases withimproved specificity. Science.2016Jan 1；351(6268):84-8)。该系统的使用极为简便，不像ZFN和TALEN那样构建繁琐，已经成为一项热门的基因编辑技术，被广泛应用于医学以及动植物育种等领域。
Inhba基因是构成TGF-β超家族成员之一ActivinA的亚基单位，其在动物卵泡的发育中具有重要作用。因而，在细胞或个体水平上对大鼠Inhba基因进行敲除或编辑，可为进一步解析大鼠卵泡发育的分子机理提供基础。
发明内容
技术问题
本发明的目的在于提供一种靶向大鼠Inhba基因的sgRNA以及利用CRISPR-Cas9系统编辑大鼠Inhba基因的方法。
技术方案
一种靶向大鼠Inhba基因的sgRNA，其特征在于,所述sgRNA命名为sgRNA-2，其核苷酸序列如SEQ ID NO.1所示；
所述的sgRNA-2在大鼠Inhba基因上位于其第二外显子的反义链上。所述的sgRNA-2其靶标序列特征符合5’-N(20)NGG-3’的排列规则，其中N(20)表示20个连续的碱基，每个N表示A或T或C或G。
所述一种靶向大鼠Inhba基因sgRNA可以利用细胞本身的CRISPR-Cas9系统在细胞水平上对大鼠Inhba基因进行有效的切割编辑或敲除应用。具体为：
1)根据sgRNA-2序列SEQ ID NO.1通过金斯瑞生物科技有限公司合成相应的单链寡核苷酸，具体序列如下：
Inhba-F2：caccggcaaaggtgatgatctccg
Inhba-R2：aaaccggagatcatcacctttgcc
用TEbuffer将Inhba-F与Inhba-R单链寡核苷酸稀释成10pmol/μl，各取10μl加入到0.2ml EP管中，然后95℃6min后，室温自然冷却退火互补；同时用BbsI酶切pX330载体后进行回收纯化，将退火互补的sgRNA-2与上述酶切回收的pX330载体连接得到 pX330-Inbha-sgRNA-2载体；
2)pX330-Inbha-sgRNA-2对大鼠Inbha基因的切割编辑
将pX330-Inbha-sgRNA-2载体转染L6细胞72h后，提取DNA，用上游引物F1与下游引物R1 组成的引物对提取的DNA进行PCR扩增(F1:5'gacttttgctgccaggatgc 3'；R1:5'cgccaccatcaccacctaat 3')；PCR扩增Inhba基因靶标序列，回收纯化；
将扩增转染pX330-Inbha-sgRNA-2的Inhba基因的Inhba基因靶标序列回收纯化产物与 pEASY-Blunt simple Cloning kit载体进行连接，将此5μl连接产物转化到Trans5a的感受态细胞中，通过T7E1酶切和克隆测序,得出重组pX330-Inbha-sgRNA-2载体利用细胞本身的 CRISPR-Cas9系统在细胞水平上对Inhba基因的切割效果。
有益效果
本发明公开了一种靶向Inhba基因的sgRNA及其用于CRISPR-Cas9系统编辑大鼠Inhba 基因的方法。该sgRNA位于大鼠Inhba基因第二外显子的反义链，其靶标序列特征符合 5’-N(20)NGG-3’的排列规则，其中N(20)表示20个连续的碱基，其中每个N表示A或T或C或G。所述的sgRNA能通过CRISPR-Cas9系统在细胞水平上对大鼠Inhba基因进行有效的编辑或敲除，并且该sgRNA序列位点没有Off-targets所述的0，1，2，3错配类型，因而在全基因组水平上是特异识别Inhba基因座的。
本发明提供的sgRNA能特异的靶向大鼠Inhba基因，并且能够以25％的效率对Inhba基因进行编辑，相比同源打靶效率(<1％)有极大的提高。同时该方法相比同源打靶、ZFN和TALEN 而言操作简单，不需要构建复杂的载体，为进一步研究大鼠Inhba基因在其卵泡发育中的功能提供了基础。
附图说明
图1为大鼠Inhba基因结构，含有三个外显子。
图2sgRNA-1在其第二外显子编码序列(SEQ ID NO 2)上的靶标位点示意图，其中下划线代表sgRNA-1位点，下虚线代表PAM位点。
图3sgRNA-2在其第二外显子编码序列(SEQ ID NO 2)上的靶标位点示意图，其中下划线代表sgRNA-2位点，下虚线代表PAM位点。
图4sgRNA-3在其第三外显子编码序列(SEQ ID NO 3)上的靶标位点示意图，其中下划线代表sgRNA-3位点，下虚线代表PAM位点。
图5为pX330-Inbha-sgRNA-1载体转染大鼠肌细胞L672h，提取细胞DNA并扩增Inhba 基因靶标序列的PCR产物经过T7E1酶切割的电泳图。
M marker，大小从下往上100，250，500，750，1000，2000kb
1转染pX330载体(阴性对照)的Inhba基因靶标序列经过T7E1酶切
2转染pX330-Inbha-sgRNA-1载体(实验组)的Inhba基因靶标序列经过T7E1酶切
图6为pX330-Inbha-sgRNA-2载体转染大鼠肌细胞L672h，提取细胞DNA并扩增Inhba 基因靶标序列的PCR产物经过T7E1酶切割的电泳图。
M marker，大小从下往上100，250，500，750，1000，2000kb
1转染pX330载体(阴性对照)的Inhba基因靶标序列经过T7E1酶切
2转染pX330-Inbha-sgRNA-2载体(实验组)的Inhba基因靶标序列经过T7E1酶切
图7为pX330-Inbha-sgRNA-3载体转染大鼠肌细胞L672h，提取细胞DNA并扩增Inhba 基因靶标序列的PCR产物经过T7E1酶切割的电泳图。
M marker，大小从下往上100，250，500，750，1000，2000kb
1转染pX330载体(阴性对照)的Inhba基因靶标序列经过T7E1酶切
2转染pX330-Inbha-sgRNA-3载体(实验组)的Inhba基因靶标序列经过T7E1酶切
图8pX330-Inbha-sgRNA-2载体转染大鼠肌细胞L672h，提取细胞DNA并扩增Inhba基因靶标序列的PCR产物进行克隆测序后有突变位点的结果
具体实施方式
本发明采用的技术方案如下：1)利用在线网站设计及选择大鼠Inbha基因的sgRNA靶标位点序列，合成相应的寡核苷酸，然后退火互补；同时用BbsI酶切pX330载体后进行回收纯化，将退火互补的sgRNA与上述酶切回收的pX330载体连接得到pX330-Inbha-sgRNA载体；2)将 pX330-Inbha-sgRNA载体转染L6细胞72h后，通过T7E1酶切和克隆测序两个方面来评价该 sgRNA对大鼠Inhba基因的编辑效率。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
(一)pX330-Inbha-sgRNA载体的构建
1、大鼠Inbha基因的sgRNA靶标位点设计
在NCBI登录号为NM_017128的序列中提取大鼠Inhba基因mRNA序列的编码区，其核苷酸序列如SEQ ID NO 4所示。使用在线软件(http://chopchop.cbu.uib.no/)(MontagueTG,Cruz JM,Gagnon JA,Church GM,Valen E.CHOPCHOP:a CRISPR/Cas9and TALEN webtool for genome editing.Nucleic Acids Res.2014Jul；42(Web Server issue):W401-7)设计sgRNA靶标位点。大鼠Inhba基因一共有三个外显子，第一外显子长度只有57bp而且又不属于该基因的编码区，因此不考虑第一外显子。分别在Inhba的第二和第三外显子上设计sgRNA靶标位点。首先考虑Off-targets指标，确保sgRNA序列位点没有Off-targets所述的0，1，2，3错配类型，以使针对Inhba基因的sgRNA靶标位点在基因组上高度特异；然后考虑Self-complementarity，选择数值为0的sgRNA位点，以防止其自身互补，阻碍其与靶标位点的结合；最后综合G含量 (40％-60％)和Efficiency(>50％)效率筛选三对sgRNA序列：其中sgRNA-1和sgRNA-2位于 Inhba基因的第二外显子，sgRNA-3位于Inhba基因的第三外显子，三对sgRNA序列如下：
sgRNA-1：agttaggtccatccttcgga
sgRNA-2：ggcaaaggtgatgatctccg
sgRNA-3：tatcatgccaactattgtga；根据sgRNA序列通过金斯瑞生物科技有限公司合成相应的单链寡核苷酸，具体序列如下：
针对sgRNA-1合成的单链寡核苷酸：
Inhba-F1：caccgagttaggtccatccttcgga
Inhba-R1：aaactccgaaggatggacctaactc
针对sgRNA-2合成的单链寡核苷酸：
Inhba-F2：caccggcaaaggtgatgatctccg
Inhba-R2：aaaccggagatcatcacctttgcc
针对sgRNA-3合成的单链寡核苷酸：
Inhba-F3：caccgtatcatgccaactattgtga
Inhba-R3：aaactcacaatagttggcatgatac
用TE buffer将Inhba-F与Inhba-R单链寡核苷酸稀释成10pmol/μl，各取10μl加入到0.2ml EP管中，然后95℃6min后，室温自然冷却。
2、pX330载体的酶切与回收纯化
酶切体系：4μg pX330载体(Addgene，货号：42335)，5μl 10X CutSmart Buffer,4μl BbsI 酶(NEB公司，货号R0539S)，加入灭菌水补足体积至50μl，37℃孵育3h，然后将酶切产物在1％的琼脂糖凝胶中通过电泳分离，切出约8kb大小的DNA条带，用琼脂糖凝胶试剂盒回收纯化DNA(ZYMO，货号：D4007)。具体步骤如下：将紫外光下切下目的DNA条带装入1.5ml离心管中，称重后加入3倍体积的ADB buffer，55℃保温10min，待琼脂糖凝胶完全融化后将其转入附柱中，10000g离心30s，倒掉流出液；加入200μl wash buffer，10000g 离心30s，倒掉流出液；重复一次；最后将吸附柱转移到一个新的1.5ml离心管中，加入10μl ddH2O，放置1min，10000g离心30s收集纯化产物。
3、Inbha-sgRNA序列与pX330载体的连接、克隆与测序
取步骤1中室温冷却的0.2ml EP管中的2μl产物，1μl pX330酶切回收产物，2μl灭菌水， 5μl solution I(Takara，货号：6013)，混匀，16℃1h。将此10μl连接产物转化到Trans5a的感受态细胞中，步骤如下：取一管的Trans5a感受态细胞(北京全式金货号：CD201-01)置于冰上7min，向其中加入10μl上述连接产物，轻弹混匀，后于冰上孵育30min；42℃热激 45s，后冰上静置2min；加入900μl无抗性的LB液体培养基，37℃200rpm震荡培养1h；用移液器吸取200μl菌液涂布在含氨苄青霉素抗性的LB培养基平板上。然后将平板倒置于37℃温箱培养12h。克隆生长出来后，挑单克隆菌落至1ml含氨苄青霉素的LB培养液中，37℃震荡培养12h，随机挑选5个克隆送去测序(金斯瑞生物科技有限公司)进一步确定阳性克隆的正确性(测序引物序列：ggactatcatatgcttaccg)，即得到pX330-Inbha-sgRNA载体。
(二)pX330-Inbha-sgRNA对大鼠Inbha基因的切割验证
1、pX330-Inbha-sgRNA转染L6细胞(国家实验细胞资源共享服务平台(北京总部)购买)
转染前一天将L6细胞铺到6孔板中，置于培养箱37℃，5％CO2条件下培养。
转染当天将重组载pX330-Inbha-sgRNA-1，pX330-Inbha-sgRNA-2，pX330-Inbha-sgRNA-3(实验组)分别和pX330载体(阴性对照组)通过脂质体3000(Invitrogen，货号： L3000150)转染L6细胞。转染的具体步骤是：在一个1.5ml EP管中加入125μl opti-MEM，然后加入3.75μl3000，混匀；在另一个1.5ml EP管中加入125μl opti-MEM，2.5μg pX330-Inbha-sgRNA载体，5μl P3000TM，混匀；将两个1.5ml EP管中的溶液混在一起，室温放置5min后，滴加到板孔中，然后置于培养箱37℃，5％CO2条件下培养。
2、转染L6细胞72h后DNA的提取
转染24h后，换新鲜全培养基，继续培养48h后收集细胞。然后用DNA提取试剂盒提取细胞DNA(Takara，货号：9765)，具体步骤是：向细胞沉淀中加入180μl的Buffer GL、20μl的Proteinase K和10μl的RNase A(10mg/ml)，于56℃水浴10min；然后向裂解液中加入200μl 100％乙醇，充分吸打混匀；将Spin Column安置于Collection Tube上，溶液移至 SpinColumn中，12,000rpm离心2min，弃滤液；将500μl的Buffer WA加入至Spin Column中，12,000rpm离心1min，弃滤液；将700μl的Buffer WB加入至Spin Column 中，12,000rpm离心1min，弃滤液；将Spin Column安置于Collection Tube上，12,000rpm 离心2min；将SpinColumn安置于新的1.5ml的离心管上，在Spin Column膜的中央处加入50μl的ElutionBuffer，室温静置5min；12,000rpm离心2min洗脱DNA。
3、Inhba基因靶标序列的PCR扩增
以步骤2提取的细胞DNA并作为模板，针对sgRNA-1和sgRNA-2，用上游引物F1与下游引物R1组成的引物对提取的DNA进行PCR扩增(F1:5'gacttttgctgccaggatgc 3'；R1:5'cgccaccatcaccacctaat 3')；回收536bp的PCR扩增产物。针对sgRNA-3，用上游引物F2与下游引物R2组成的引物对提取的DNA进行PCR扩增(F2:5'tgctcctgggcaagaagaag 3'；R2: 5'gacctggcaactctaggagc 3')；回收524bp的PCR扩增产物。PCR反应体系如下：25μlPrimerSTAR Max DNAPolymerase(TaKaRa，货号：R045A)，50ng DNA，上游引物F 0.3uM，下游引物R 0.3uM，加超纯水至50μl。PCR反应程序95℃预变性2min，1个循环；98℃10s, 60℃15s,72℃15s，30个循环；72℃后延伸3min。PCR产物用琼脂糖凝胶回收试剂盒进行回收纯化(ZYMO，货号：D4007)，具体步骤同上。
4Inhba基因靶标序列PCR产物的酶切
利用T7E1(NEB，货号：M0263S)将步骤3得到的PCR回收纯化产物先进行PCR梯度变性，然后再进行酶切。PCR梯度变性程序：200ng PCR回收产物，1.1μl T7E1buffer，灭菌水补齐到10.5μl，然后进行PCR梯度变性，程序为：95℃变性5min；95-85℃，每秒降低 2℃；85-25℃，每秒降低0.1℃。酶切反应体系如下：10.5μl上述产物，0.5μl T7E1，37℃ 30min，然后在2％的琼脂糖进行电泳，由图5和图7可以看到转染pX330载体组(阴性对照) 的Inhba基因靶标序列经过T7E1酶切后没有出现条带与预期相符，而转染 pX330-Inbha-sgRNA-1和pX330-Inbha-sgRNA-3(实验组)的Inhba基因靶标序列经过T7E1酶切后也未出现预期的切割条带，表明经过软件选中的两条sgRNA-1和sgRNA-3无效。而由图6可以看到转染pX330载体组(阴性对照)的Inhba基因靶标序列经过T7E1酶切后没有出现条带与预期相符，转染pX330-Inbha-sgRNA-2(实验组)的Inhba基因靶标序列经过T7E1 酶切后，536bp的产物被切割为大约386bp和150bp，出现预期的切割条带，表明sgRNA-2 有效。
5Inhba基因靶标序列PCR产物的克隆测序(切割编辑效果)
将扩增转染pX330-Inbha-sgRNA-2(实验组)的Inhba基因的PCR回收纯化产物与pEASY-Blunt simple Cloning kit载体(北京全式金，货号：CB111-01)进行连接，步骤为在0.2mlEP管中加入1μlpEASY-Blunt载体，PCR产物4μl，25℃反应10min。将此5μl连接产物转化到Trans5a的感受态细胞中，步骤同上。随机挑选20个克隆送去测序(金斯瑞生物科技有限公司)，计算有序列突变的克隆数占总体克隆数的比例，从而估算pX330-Inhba-sgRNA-2载体对 Inhba基因的切割效率。结果发现5个克隆在预期切割位点附近出现突变(图8)，即重组 pX330-Inbha-sgRNA-2载体利用细胞本身的CRISPR-Cas9系统在细胞水平上对Inhba基因的切割效率为25％。
>SEQ ID NO 1
ggcaaaggtgatgatctccg
>SEQ ID NO 2
atgcccttgctttggctgagaggatttctgttggcaagttgctggattatagtgaggagttcccccaccccaggatccgaggggcacggcgcagccccggactgccc gtcctgtgcgctggccacccttccgaaggatggacctaactctcagccagagatggtagaggctgtcaagaagcacatcttaaacatgctgcacttgaagaagagacccgatgtcacccagccggtacccaaggcggcgcttctcaacgcgatcagaaagcttcatgtgggtaaagtgggggaaaacgggtatgtggagatagaggacgaca ttggcaggagggccgaaatgaatgaactcatggagcagacctcggagatcatcacctttgccgagtcag
>SEQ ID NO 3
gcacagccaggaagacactgcattttgagatttccaaggaaggcagtgacctgtcagtcgtggagcgtgcagaagtctggctcttcctgaaagtccccaaggccaac aggaccaggaccaaagtcaccatccgtctgtttcagcagcagaagcatccacagggcagcttggacatgggggatgaggccgaggaaatgggcttgaaggggga gaggagtgaactgttgctatcagagaaagtggtagatgctcggaagagtacttggcacatcttcccagtgtctagcagcatccagcgcctgctggaccaggggaaga gttccctggatgtgcggattgcttgtgaacagtgccaggagagcggtgccagcctagtgctcctgggcaagaagaagaagaaagaggtggatggagacgggaag aagaaagacggaagtgacggagggctggaagaggaaaaagaacagtcacacagacctttcctcatgctgcaggctaggcagtctgaagaccatcctcaccgcaggcgtaggcggggcttggagtgtgatggcaaggtcaacatttgctgtaagaaacagttctttgtcagcttcaaggatattggctggaatgactggatcattgctccctctg gctatcatgccaactattgtgagggtgagtgcccgagccacatagcaggcacctctgggtcctcactctccttccactcaacagtcattaaccactaccgcatgaggggtcacagcccctttgccaaccttaagtcatgctgtgtgcccaccaagctgagacccatgtccatgctgtattatgatgatggtcaaaacattatcaaaaaggacattcagaa catgattgtggaggagtgtggctgctcctag
>SEQ ID NO 4
atgcccttgctttggctgagaggatttctgttggcaagttgctggattatagtgaggagttcccccaccccaggatccgaggggcacggcgcagccccggactgcccgtcctgt gcgctggccacccttccgaaggatggacctaactctcagccagagatggtagaggctgtcaagaagcacatcttaaacatgctgcacttgaagaagagacccgatgtcacccagccggtacccaaggcggcgcttctcaacgcgatcagaaagcttcatgtgggtaaagtgggggaaaacgggtatgtggagatagaggacgacattggcaggagggccga aatgaatgaactcatggagcagacctcggagatcatcacctttgccgagtcaggcacagccaggaagacactgcattttgagatttccaaggaaggcagtgacctgtcagtcgtggagcgtgcagaagtctggctcttcctgaaagtccccaaggccaacaggaccaggaccaaagtcaccatccgtctgtttcagcagcagaagcatccacagggcagcttgg acatgggggatgaggccgaggaaatgggcttgaagggggagaggagtgaactgttgctatcagagaaagtggtagatgctcggaagagtacttggcacatcttcccagtgtctagcagcatccagcgcctgctggaccaggggaagagttccctggatgtgcggattgcttgtgaacagtgccaggagagcggtgccagcctagtgctcctgggcaagaaga agaagaaagaggtggatggagacgggaagaagaaagacggaagtgacggagggctggaagaggaaaaagaacagtcacacagacctttcctcatgctgcaggctaggcagtctgaagaccatcctcaccgcaggcgtaggcggggcttggagtgtgatggcaaggtcaacatttgctgtaagaaacagttctttgtcagcttcaaggatattggctggaatg actggatcattgctccctctggctatcatgccaactattgtgagggtgagtgcccgagccacatagcaggcacctctgggtcctcactctccttccactcaacagtcattaaccactaccgcatgaggggtcacagcccctttgccaaccttaagtcatgctgtgtgcccaccaagctgagacccatgtccatgctgtattatgatgatggtcaaaacattatcaaaaagg acattcagaacatgattgtggaggagtgtggctgctcctag。
序列表
<110> 江苏省农业科学院
<120> 一种靶向大鼠Inhba基因的sgRNA及其应用
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(20)
<400> 1
ggcaaaggtg atgatctccg 20
<210> 2
<211> 388
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(388)
<400> 2
atgcccttgc tttggctgag aggatttctg ttggcaagtt gctggattat agtgaggagt 60
tcccccaccc caggatccga ggggcacggc gcagccccgg actgcccgtc ctgtgcgctg 120
gccacccttc cgaaggatgg acctaactct cagccagaga tggtagaggc tgtcaagaag 180
cacatcttaa acatgctgca cttgaagaag agacccgatg tcacccagcc ggtacccaag 240
gcggcgcttc tcaacgcgat cagaaagctt catgtgggta aagtggggga aaacgggtat 300
gtggagatag aggacgacat tggcaggagg gccgaaatga atgaactcat ggagcagacc 360
tcggagatca tcacctttgc cgagtcag 388
<210> 3
<211> 887
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(887)
<400> 3
gcacagccag gaagacactg cattttgaga tttccaagga aggcagtgac ctgtcagtcg 60
tggagcgtgc agaagtctgg ctcttcctga aagtccccaa ggccaacagg accaggacca 120
aagtcaccat ccgtctgttt cagcagcaga agcatccaca gggcagcttg gacatggggg 180
atgaggccga ggaaatgggc ttgaaggggg agaggagtga actgttgcta tcagagaaag 240
tggtagatgc tcggaagagt acttggcaca tcttcccagt gtctagcagc atccagcgcc 300
tgctggacca ggggaagagt tccctggatg tgcggattgc ttgtgaacag tgccaggaga 360
gcggtgccag cctagtgctc ctgggcaaga agaagaagaa agaggtggat ggagacggga 420
agaagaaaga cggaagtgac ggagggctgg aagaggaaaa agaacagtca cacagacctt 480
tcctcatgct gcaggctagg cagtctgaag accatcctca ccgcaggcgt aggcggggct 540
tggagtgtga tggcaaggtc aacatttgct gtaagaaaca gttctttgtc agcttcaagg 600
atattggctg gaatgactgg atcattgctc cctctggcta tcatgccaac tattgtgagg 660
gtgagtgccc gagccacata gcaggcacct ctgggtcctc actctccttc cactcaacag 720
tcattaacca ctaccgcatg aggggtcaca gcccctttgc caaccttaag tcatgctgtg 780
tgcccaccaa gctgagaccc atgtccatgc tgtattatga tgatggtcaa aacattatca 840
aaaaggacat tcagaacatg attgtggagg agtgtggctg ctcctag 887
<210> 4
<211> 1275
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> mRNA
<222> (1)..(1275)
<400> 4
atgcccttgc tttggctgag aggatttctg ttggcaagtt gctggattat agtgaggagt 60
tcccccaccc caggatccga ggggcacggc gcagccccgg actgcccgtc ctgtgcgctg 120
gccacccttc cgaaggatgg acctaactct cagccagaga tggtagaggc tgtcaagaag 180
cacatcttaa acatgctgca cttgaagaag agacccgatg tcacccagcc ggtacccaag 240
gcggcgcttc tcaacgcgat cagaaagctt catgtgggta aagtggggga aaacgggtat 300
gtggagatag aggacgacat tggcaggagg gccgaaatga atgaactcat ggagcagacc 360
tcggagatca tcacctttgc cgagtcaggc acagccagga agacactgca ttttgagatt 420
tccaaggaag gcagtgacct gtcagtcgtg gagcgtgcag aagtctggct cttcctgaaa 480
gtccccaagg ccaacaggac caggaccaaa gtcaccatcc gtctgtttca gcagcagaag 540
catccacagg gcagcttgga catgggggat gaggccgagg aaatgggctt gaagggggag 600
aggagtgaac tgttgctatc agagaaagtg gtagatgctc ggaagagtac ttggcacatc 660
ttcccagtgt ctagcagcat ccagcgcctg ctggaccagg ggaagagttc cctggatgtg 720
cggattgctt gtgaacagtg ccaggagagc ggtgccagcc tagtgctcct gggcaagaag 780
aagaagaaag aggtggatgg agacgggaag aagaaagacg gaagtgacgg agggctggaa 840
gaggaaaaag aacagtcaca cagacctttc ctcatgctgc aggctaggca gtctgaagac 900
catcctcacc gcaggcgtag gcggggcttg gagtgtgatg gcaaggtcaa catttgctgt 960
aagaaacagt tctttgtcag cttcaaggat attggctgga atgactggat cattgctccc 1020
tctggctatc atgccaacta ttgtgagggt gagtgcccga gccacatagc aggcacctct 1080
gggtcctcac tctccttcca ctcaacagtc attaaccact accgcatgag gggtcacagc 1140
ccctttgcca accttaagtc atgctgtgtg cccaccaagc tgagacccat gtccatgctg 1200
tattatgatg atggtcaaaa cattatcaaa aaggacattc agaacatgat tgtggaggag 1260
tgtggctgct cctag 1275
<210> 5
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(20)
<400> 5
agttaggtcc atccttcgga 20
<210> 6
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(20)
<400> 6
tatcatgcca actattgtga 20
<210> 7
<211> 25
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(25)
<400> 7
caccgagtta ggtccatcct tcgga 25
<210> 8
<211> 25
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(25)
<400> 8
aaactccgaa ggatggacct aactc 25
<210> 9
<211> 24
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(24)
<400> 9
caccggcaaa ggtgatgatc tccg 24
<210> 10
<211> 24
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(24)
<400> 10
aaaccggaga tcatcacctt tgcc 24
<210> 11
<211> 25
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(25)
<400> 11
caccgtatca tgccaactat tgtga 25
<210> 12
<211> 25
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> exon
<222> (1)..(25)
<400> 12
aaactcacaa tagttggcat gatac 25
<210> 13
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 13
gacttttgct gccaggatgc 20
<210> 14
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 14
cgccaccatc accacctaat 20
<210> 15
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 15
tgctcctggg caagaagaag 20
<210> 16
<211> 20
<212> DNA
<213> 大鼠(2 Ambystoma laterale x Ambystoma jeffersonianum)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 16
gacctggcaa ctctaggagc 20
<210> 17
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<220>
<221> primer_bind
<222> (1)..(20)
<400> 17
ggactatcat atgcttaccg 20
Claims (6)
1.一种靶向大鼠Inhba基因的sgRNA，其特征在于，所述sgRNA命名为sgRNA-2，其核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的一种靶向大鼠Inhba基因的sgRNA，其特征在于,所述的sgRNA-2在大鼠Inhba基因上位于其第二外显子的反义链上。
3.根据权利要求1或2所述的一种靶向大鼠Inhba基因的sgRNA，其特征在于,所述的sgRNA-2其靶标序列特征符合5’-N(20)NGG-3’的排列规则，其中N(20)表示20个连续的碱基，每个N表示A或T或C或G。
4.权利要求1-3之一所述一种靶向大鼠Inhba基因sgRNA的应用。
5.根据权利要求4所述的应用，其特征在于，是指所述的sgRNA通过CRISPR-Cas9系统在细胞水平上对大鼠Inhba基因进行有效的编辑或敲除应用。
6.根据权利要求4或5所述的应用，其特征在于，
1)根据sgRNA-2序列SEQ ID NO.1通过金斯瑞生物科技有限公司合成相应的单链寡核苷酸，具体序列如下：
Inhba-F2：caccggcaaaggtgatgatctccg
Inhba-R2：aaaccggagatcatcacctttgcc
用TE buffer将Inhba-F与Inhba-R单链寡核苷酸稀释成10pmol/μl，各取10μl加入到0.2ml EP管中，然后95℃6min后，室温自然冷却退火互补；同时用BbsI酶切pX330载体后进行回收纯化，将退火互补的sgRNA-2与上述酶切回收的pX330载体连接得到pX330-Inbha-sgRNA-2载体；
2)pX330-Inbha-sgRNA-2对大鼠Inbha基因的切割
将pX330-Inbha-sgRNA-2载体转染L6细胞72h后，提取DNA，用上游引物F1与下游引物R1组成的引物对提取的DNA进行PCR扩增，PCR扩增Inhba基因靶标序列，回收纯化；
F1:5'gacttttgctgccaggatgc 3'；
R1:5'cgccaccatcaccacctaat 3'；
将扩增转染pX330-Inbha-sgRNA-2的Inhba基因靶标序列回收纯化产物与pEASY-Bluntsimple Cloningkit载体进行连接，将此5ul连接产物转化到Trans5a的感受态细胞中，通过T7E1酶切和克隆测序,得出重组pX330-Inbha-sgRNA-2载体对Inhba基因的切割效果。
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