CN107058320B - Il7r基因缺失斑马鱼突变体的制备及其应用 - Google Patents
Il7r基因缺失斑马鱼突变体的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种IL7R基因缺失斑马鱼突变体及其制备方法,IL7R基因缺失斑马鱼突变体的构建是通过CRISPR/Cas9技术实现的。另外,本发明还公开了IL7R基因缺失斑马鱼突变体的应用,本发明的突变体模型可用于研究IL7R在神经系统发育及髓鞘形成中的作用。
Description
技术领域
本发明属于分子生物学领域,涉及一种基因敲除的斑马鱼突变体,具体涉及IL7R基因缺失斑马鱼突变体及其制备方法和应用。
背景技术
白细胞介素7受体(interleukin 7receptor,IL7R)属于Ⅰ型细胞因子受体家族成员的跨膜受体,由α链(IL7Rα,CD127)和γ链(γc,CD132)组成,α链是IL7R的特异性组分;γ链是IL2、IL4、IL7、IL9、IL15、IL21等细胞因子受体的共同组分,也被称为共有γ链,是启动IL7R下游信号表达的必需成分。生理情况下,IL7R是淋巴细胞增殖和分化的必需关键因子;病理情况下,IL7R功能缺陷可导致重症联合免疫缺陷病,而IL7R获得性突变可引起急性T淋巴细胞白血病等。
成簇规律间隔短回文重复系统(Clustered regularly interspaced shortpalindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas),CRISPR/Cas)系统是细菌或古细菌在长期演化过程中形成的抵御外来遗传物质的一种获得性免疫防御机制,能够识别自身和外源入侵DNA片段。CRISPR/Cas系统有3种类型:Ⅰ型、Ⅱ型和Ⅲ型,其中II型CRISPR/Cas系统只需要一个Cas9核酸内切酶切割DNA双链,即CRISPR/Cas9系统。在向导RNA(single guide,sgRNA)指导下,Cas9内切酶像剪刀一样对特定基因、特定位点进行靶向性剪切,形成双链DNA缺口,细胞可以通过同源重组修复或者非同源性末端连接对断裂DNA进行修复,从而实现对基因的编辑。作为一种新兴基因组编辑技术,CRISPR/Cas9系统具有实验简单、操作容易和节省时间等优势。利用CRISPR/Cas9技术在酵母、拟南芥、小鼠、大鼠、果蝇、青蛙等不同物种都成功进行了基因定向改造。在斑马鱼研究领域,CRISPR/Cas9系统也被广泛应用于精确和靶向性的基因编辑。在单细胞胚胎阶段通过注射sgRNA和Cas9 mRNA能够快速、高效获得基因敲除斑马鱼突变体,并且可以遗传给子代,这大大拓展了在斑马鱼中进行基因功能缺失的研究。
申请人先在幼鱼脱髓鞘模型上以基因芯片进行了表达谱分析,在获得的差异表达基因中,申请人发现,IL7R表达显著降低,提示IL7R可能与神经系统的发育有密切关系。斑马鱼的IL7R和人类IL7R的相似度为73%,利用IL7R基因敲除突变体进行研究是一个有效途径,然而,目前国内外尚无报道。申请人认为,有必要构建IL7R基因敲除斑马鱼突变体,并以此为模型对IL7R在神经系统发育及髓鞘形成中的作用进行在体实验研究。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明的一个目的在于提供一种sgRNA片段以及使用该sgRNA片段构建IL7R基因缺失斑马鱼突变体的方法。为此,发明人进行了大量的实验,在众多sgRNA中筛选出活性最高的一种sgRNA并将其用于突变体的构建中,从而获得了稳定遗传的IL7R基因缺失斑马鱼突变体。
为了实现上述目的,本发明采用了如下技术方案:
根据本发明的一个方面,本发明提供了一种sgRNA片段,所述sgRNA的DNA序列如5-’ACTCCACTCACTCCAGTCACCGG-3’所示,其中3’端的CGG为PAM识别位点。
本发明提供了一种基因打靶试剂盒,所述试剂盒包括前面所述的sgRNA。使用该试剂盒可以用于IL7R基因表达的沉默。
本发明提供了一种基因敲除方法,所述方法的步骤如下:利用前面所述的sgRNA片段识别目的基因上的双链上的不同靶位点,与一种核酸酶结合并通过识别靶位点处PAM序列,引导核酸酶结合到目的基因靶位点处,并开始进行随机剪切,随后从切口处脱落,形成DSB缺口,随后细胞通过非同源末端连接修复机制修复目的基因双链,造成移码突变,最终目的基因被敲除。
进一步,所述sgRNA片段与核酸酶发挥作用的数量为一个或以上。
根据本发明的又一个方面,本发明提供了一种IL7R基因缺失斑马鱼突变体的制备方法,所述制备方法包括:
(1)将前面所述的sgRNA和Cas9 mRNA共同导入斑马鱼中;
(2)培养获得稳定遗传的IL7R基因缺失斑马鱼突变体。
在本发明的具体实施方案中,所述sgRNA的获得步骤如下:
(1)确定IL7R基因敲除的靶位点;
(2)根据步骤(1)确定的靶位点序列设计扩增引物,PCR获得sgRNA的体外转录的模板;
(3)根据步骤(2)获得的模板进行体外转录。
作为本发明的具体实施例,所述sgRNA的获得步骤如下:
(1)查询斑马鱼IL7R基因的基因组DNA序列及其功能结构域,根据CRISPR/Cas9敲除原理,设计一对IL7R基因的靶位点。
(2)PCR法获得sgRNA的体外转录的模板,正向引物序列为:5’TAATACGACTCACTATAGACTCCACTCACTCCAGTCACgttttagagctagaaatagc-3’(前18位碱基序列是T7启动子,19-38位碱基序列是sgRNA靶序列,小写字母表示的碱基序列为sgRNA上游骨架);反向引物序列为25bp sgRNA下游骨架,序列为5’-AAAAAAAGCACCGACTCGGTGCCAC-3’,按照下列PCR条件进行扩增:预变性95℃3min;变性95℃30s,退火54℃30s,延伸72℃20s进行35个循环,再72℃10min;PCR产物回收纯化。
(3)以RNA体外转录试剂盒(MAXIscript T7,Ambion,CA,USA)进行体外转录。
在本发明的具体实施方案中,所述Cas9 mRNA是按照以下步骤获得的:
以T7驱动的人源化的Cas9编码序列为模板,用T7RNA聚合酶进行Cas9体外转录(mMESSAGE mMACHINE T7,Ambion),然后戴帽并加尾(polyA)。RNA经无水乙醇法沉淀,用DEPC处理过的超纯水重悬。
作为本发明的具体实施例,sgRNA和Cas9 mRNA共同导入斑马鱼中,包括:将sgRNA8和Cas9 mRNA混合,sgRNA终浓度为50ng/μL、Cas9 mRNA终浓度为250ng/μL,在斑马鱼胚胎1-4细胞期进行显微注射,注射量为1nL。
在本发明的具体实施方案中,培养获得稳定遗传的IL7R基因缺失斑马鱼突变体,包括:
(1)对经过注射的斑马鱼进行基因型鉴定,确定IL7R基因敲除的初建者F0;
(2)IL7R基因敲除的初建者F0个体进行自交获得F1;
(3)利用基因型鉴定的方法确定IL7R基因敲除的F1;
(4)从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代;
(5)鉴定为F2代中IL7R基因敲除的纯合子即为稳定遗传的IL7R基因缺失斑马鱼突变体。
根据本发明的另一方面,本发明提供了前面所述的IL7R基因缺失斑马鱼突变体在制备研究IL7R在神经系统发育中的作用的材料中的应用。
本发明的IL7R基因缺失斑马鱼突变体的构建动机在于:在斑马鱼幼鱼脱髓鞘模型上以基因芯片进行了表达谱分析,在获得的差异表达基因中,发现IL7R基因表达显著降低,提示IL7R基因可能与神经系统的发育有密切关系。因此有必要构建IL7R基因敲除斑马鱼突变体,并以此为模型对IL7R在神经系统发育及髓鞘形成的作用进行在体实验研究。
本发明的有益效果和优点在于:
本发明基于IL7R基因可能与神经系统的发育有密切关系的实验结果构建了IL7R基因缺失斑马鱼突变体,该突变体为国内外首例。
本发明的构建的IL7R基因缺失斑马鱼突变体可稳定遗传,方便对IL7R基因在神经系统发育及髓鞘形成的作用进行在体实验研究。
附图说明
图1显示利用Western Blot检测斑马鱼纯合突变体IL7R-/-中IL7R蛋白表达情况;
图2显示野生型斑马鱼和斑马鱼突变体中IL7R基因序列反向对比图;
图3显示野生型斑马鱼和斑马鱼突变体中IL7R基因靶位点附近序列缺失对比图;
图4是cDNA基因芯片热图显示的髓鞘脱失斑马鱼模型的差异表达基因谱;
图5显示利用qRT-PCR检测髓鞘脱失斑马鱼模型中髓鞘碱性蛋白mbp mRNA的表达情况;
图6显示利用qRT-PCR检测髓鞘脱失斑马鱼模型中IL7R基因mRNA的表达情况;
图7显示利用Western Blot检测髓鞘脱失斑马鱼模型中IL7R蛋白的表达情况;
图8显示斑马鱼纯合突变体IL7R-/-鉴定,其中,A:电泳图、B:测序图;
图9显示斑马鱼纯合突变体IL7R-/-检测脱靶效应的测序图。
具体实施方式
下面通过实施例进一步说明本发明。应该理解的是,本发明的实施例是用于说明本发明而不是对本发明的限制。根据本发明的实质对本发明进行的简单改进都属于本发明要求保护的范围。
下述实施例中所使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。
实施例1筛选髓鞘脱失斑马鱼模型差异表达基因
1、步骤
利用基因芯片(Affymetrix GeneChip Zeb Gene 1.0ST Array),对髓鞘脱失斑马鱼模型(构建方法同专利文献:ZL2014101092570)的基因表达谱进行检测和分析。差异表达基因判定标准为:≥2倍为上调;≤0.5倍为下调。芯片分析发现1364个差异基因,包括271个上调基因,1093个下调基因(图4)。在众多下调基因中,发现IL7R表达显著下调(foldchange=0.44,*p=0.0061888)。以实时荧光定量PCR(qRT-PCR)及蛋白印迹实验(Westernblot,WB)进行验证。验证方法为:实验组为5dpf Tg(mbp:nfsB-egfp),以0.2%DMSO-5mM甲硝唑(mitronidazole,Mtz)处理5天至10dpf;对照组(Control,Ctrl)为5dpf Tg(mbp:nfsB-egfp),以0.2%DMSO处理至10dpf。首先,在5、6、8和10dpf分别提取对照组及Mtz处理组的RNA,检测了髓鞘碱性蛋白(myelin basic protein,mbp)mRNA的表达,以验证髓鞘脱失。mbp(GenBank NM_001271459)引物序列为:正向序列5’-GGGCAGAAAGAAGAAGGC-3’;,反向序列5’-CGGGTGGAAGAGTGGTG-3’。结果显示,Mtz处理组的mbp mRNA的表达水平随药物作用延长而下降,在8dpf和10dpf显著低于对照组(图5;Student’s t test,*P<0.05)。第二,以qRT-PCR方法对5、6、8和10dpf IL7R mRNA的表达量进行定量检测,IL7R(GenBank NM_001113507)引物序列为:正向序列,5’-TTACACCAAACATCCCACA-3’;反向序列,5’-TAGGCTTCTTCTTTACATTC-3’。实验结果显示,Mtz处理组IL7R mRNA表达量亦随药物作用时间而下降,在8dpf和10dpf显著低于对照组(图6;Student’s t test,*P<0.05)。第三,在10dpf提取对照组和Mtz处理组的蛋白,检测IL7R蛋白的表达情况。10dpf Mtz处理组IL7R蛋白的表达量低于对照组(图7)。上述结果证明,在斑马鱼髓鞘脱失时,IL7R的表达明显下调。
实施例2 sgRNA的筛选
1、实验动物
按标准化方案养殖野生型斑马鱼(TU品系),水温28.5℃,光照/黑暗周期为14h/10h,成体斑马鱼产卵后收集胚胎,养殖于E3孵化液,以受精的小时数(hours post-fertilization,hpf)或受精的天数(days post-fertilization,dpf)表示胚胎和幼鱼的发育阶段。
2、CRISPR/Cas9基因敲除靶位点设计
在http://asia.ensembl.org上查询斑马鱼IL7R基因的基因组DNA序列及其功能结构域,根据CRISPR/Cas9敲除原理,在http://crispr.mit.edu/上设计一对IL7R基因的靶位点,靶位点包含20个碱基,靶点的选择标准为:5’-GG-(N)20-NGG-3’;其中5’端的GG二核苷酸是T7启动子的一部分,靶位点的3’端是NGG(N为任意碱基)。
3、sgRNA活性检测
3.1步骤
针对IL7R基因外显子3、4、5和6共设计合成了12个sgRNA,两个sgRNA一组,与Cas9mRNA进行共注射。提取胚胎DNA选择直接测序法进行sgRNA活性分析。sgRNA序列如表1所示。sgRNA体外合成步骤、注射步骤、胚胎DNA提取步骤同后面实施例3中提到的相同。
3.2结果
结果如表1所示,第四组(sgRNA7和8)的活性最高,为5.9%。测序结果表明,可导致4个碱基缺失突变的是sgRNA8。因此,以sgRNA8和Cas9 mRNA共注射,制备IL7R基因缺失斑马鱼突变体。
表1sg RNA序列及活性测试
实施例3斑马鱼胚胎的显微注射及靶位点有效性测定
1、sgRNA体外合成
1.1PCR法获得sgRNA的体外转录的模板
正向引物序列为:5’-TAATACGACTCACTATAGACTCCACTCACTCCAGTCACgttttagagctagaaatagc-3’(前18位碱基序列是T7启动子,19-38位碱基序列是sgRNA靶序列,小写字母表示的碱基序列为sgRNA上游骨架);反向引物序列为25bp sgRNA下游骨架,序列为5’-AAAAAAAGCACCGACTCGGTGCCAC-3’,按照下列PCR条件进行扩增:预变性95℃3min;变性95℃30s,退火54℃30s,延伸72℃20s进行35个循环,再72℃10min;
1.2PCR产物回收纯化;
1.3以RNA体外转录试剂盒(MAXIscript T7,Ambion,CA,USA)进行体外转录,合成特异性sgRNA。
2、Cas9 mRNA体外转录
以T7驱动的人源化的Cas9编码序列为模板,用T7RNA聚合酶进行Cas9体外转录(mMESSAGE mMACHINE T7,Ambion),然后戴帽并加尾(polyA)。RNA经无水乙醇法沉淀,用DEPC处理过的超纯水重悬,-80℃储存备用。
3、斑马鱼胚胎的显微注射及靶位点有效性测定
将sgRNA和Cas9 mRNA混合(sgRNA终浓度为50ng/μL、Cas9 mRNA终浓度为250ng/μL),在斑马鱼胚胎1-4细胞期进行显微注射,注射量为1nl。注射过的受精卵放置于E3培养液中(5mmol/L NaCl,0.17mmol/L KCl,0.33mmol/L CaCl2,0.33mmol/L MgSO4,pH 7.2),28.5℃条件下孵化。待胚胎发育至7hpf时,挑选5枚发育正常的早期胚胎,用一步法PCR痕量基因型鉴定试剂盒(YSY Biotech,China)制备基因组DNA模板,以引物对IL7R-Exon5F(5’-GGTTTGAACACCGTCATGATT-3’)和IL7R-Exon5R(5’-AAGTGGGATTTGAAACAACGA-3’)进行PCR扩增,反应条件为:预变性95℃3min;变性95℃30s,退火60℃30s,延伸72℃30s进行35个循环;72℃10min,PCR产物克隆至pGEM-T Easy载体。以引物对T7和SP6对转化子进行扩增,将PCR产物送测序,测序引物为IL7R-Exon5F,检测靶位点的有效性。
实施例4 IL7R基因敲除初建者F0的制备
按照实施例3的方法对斑马鱼胚胎进行显微注射,60dpf后在IL7R基因敲除的F0代中随机挑选4尾,用无菌剪剪取尾鳍,体积约1mm3即1μL,制备基因组DNA模板,按实施例3的方法对F0进行鉴定,确定初建者F0。
实施例5 F1突变体筛选
1、步骤
收取初建者F0自交的胚胎获得F1,将F1按常规饲养至60dpf以上,选取仔鱼,剪取部分尾鳍组织进行基因型鉴定,确定此突变是否可以遗传到Fl代。
2、结果
将斑马鱼突变体的F1代养大至2-3月龄,再分别对每条Fl代斑马鱼进行剪尾,筛选Fl代突变体。图2为野生型和缺失型基因序列反向对比图,该图说明在外显子5处缺失4个碱基,存在移码突变。图3为靶位点附近序列缺失比对图,显示敲除成功,有突变型鱼出现。
实施例6获得可遗传的F2代斑马鱼纯合突变体IL7R-/-
1、步骤
从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代,放置于28.5℃培养,于4dpf取部分胚胎,每个胚胎单独提取基因组DNA,再以引物对IL7R-4bp wt F1、IL7R-4bpmut F1和IL7R-Exon5R进行PCR扩增。引物序列如下:
IL7R-4bp wt F1(鉴定野生型):5’-AACTATTTTGCCGGTGAC-3’;
IL7R-4bp mut F1(鉴定突变体):5’-AACTATTTTGCCGGTGGA-3’;
IL7R-Exon5R(共用反向引物)5’-AAGTGGGATTTGAAACAACGA-3’;
F2代中,野生型个体在引物对IL7R-4bp wt F1/IL7R-Exon5R扩增下有条带,IL7R-4bp mut F1/IL7R-Exon5R扩增下无条带,杂合突变体在引物对IL7R-4bp wt F1/IL7R-Exon5R和IL7R-4bp mut F1/IL7R-Exon5R R扩增下均有条带,纯合突变体在IL7R-4bp wtF1/IL7R-Exon5R扩增下无条带,而在IL7R-4bp mut F1/IL7R-Exon5R扩增下有条带。通过PCR条带分析将电泳条带鉴定为纯合突变的鱼以IL7R-Exon5F为引物进行测序来做进一步鉴定。若测序峰图为单一条带且基因缺失4个碱基,则确定为纯合子。如检测结果证明存在纯合子,则养大后再单条剪尾鉴定,检测是否存在脱靶效应。
针对前10个脱靶位点设计引物如下:
表2针对前10个脱靶位点设计的引物
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应。反应条件为:预变性95℃3min,其中变性95℃20s,退火60℃20s,延伸72℃20s进行35个循环,再72℃10min;待反应结束后,离心PCR产物,取1μL样品点样于2.0%琼脂糖凝胶上进行电泳,凝胶成像系统拍摄结果;将PCR产物割胶回收并进行纯化送测序,测序引物为off-target n F/R,检测其是否存在脱靶。
2、结果
2.1鉴定斑马鱼突变体F2代纯合子IL7R-/-
结果如图8所示,1、2、3、4号鱼为纯合子,5、7、8号鱼为杂合子,6号鱼为野生型(图8A)。通过PCR条带分析将电泳条带鉴定为纯合突变的鱼以IL7R-Exon5F为引物进行测序来做进一步鉴定。测序峰图为单一条带且基因缺失4个碱基,则可以确定为纯合子IL7R-/-(图8B)。将检验结果证实的纯合子常规养殖至成体后再单条剪尾鉴定。
2.2F2突变体纯合子检测脱靶效应
在http://crispr.mit.edu/上面输入sgRNA序列,并选取前10个潜在的脱靶位点,设计引物鉴定是否存在脱靶效应,图9表明,在靶位点处突变体与野生型序列一致,且无套峰,说明在此纯合突变体IL7R-/-中不存在脱靶效应。
实施例7 IL7R基因缺失斑马鱼突变体在蛋白水平的鉴定
蛋白印迹
对F2代IL7R-/-进行该基因缺失型斑马鱼纯系遗传,得到F3代IL7R-/-。于4dpf,随机取野生型与IL7R-/-突变体斑马鱼各30条,按常规方法提取蛋白质并定量,进行蛋白印迹实验。所用抗体为:anti-IL7R多克隆抗体(1:400,sc-662;Santa Cruz Biotechnology Inc.,TX,USA)。anti-β-Actin单克隆抗体(1:3000,sc-47778;Santa Cruz Biotechnology Inc.)为内参照。
结果:以蛋白印迹方法检测IL7R-/-中IL7R蛋白表达水平,图1表明,与野生型相比,纯合突变体的IL7R无表达,说明,通过CRISPR/Cas9技术成功构建了IL7R缺失型斑马鱼突变体。
虽然以上仅描述了本发明的具体实施方式范例,但是本领域的技术人员应当理解,这些仅是举例说明,本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下,可以对这些实施方式做出多种变更或修改,但这些变更或修改均落入本发明的保护范围。
SEQUENCE LISTING
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<213> 人工序列
<400> 24
tttatgtgct ccacctgtc 19
<210> 25
<211> 19
<212> DNA
<213> 人工序列
<400> 25
aaaagttctc cagggtgtc 19
<210> 26
<211> 19
<212> DNA
<213> 人工序列
<400> 26
aagtggcttt gtcattgct 19
<210> 27
<211> 23
<212> DNA
<213> 人工序列
<400> 27
atcctggaga caccaaaatc tta 23
<210> 28
<211> 20
<212> DNA
<213> 人工序列
<400> 28
cgtgaacaac cagccaaaca 20
<210> 29
<211> 23
<212> DNA
<213> 人工序列
<400> 29
ggctgatttc tcacctatct cct 23
<210> 30
<211> 21
<212> DNA
<213> 人工序列
<400> 30
tttcagtgct gagtggtgtg a 21
<210> 31
<211> 19
<212> DNA
<213> 人工序列
<400> 31
aagtggaaga tatgttagc 19
<210> 32
<211> 19
<212> DNA
<213> 人工序列
<400> 32
attttcttac gactgaact 19
<210> 33
<211> 19
<212> DNA
<213> 人工序列
<400> 33
cccgaggtta tagtcttaa 19
<210> 34
<211> 19
<212> DNA
<213> 人工序列
<400> 34
aaaatctgca taggtcaaa 19
<210> 35
<211> 17
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<213> 人工序列
<400> 35
agcgttaaaa cccaact 17
<210> 36
<211> 18
<212> DNA
<213> 人工序列
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cttcacatct gtcctccc 18
<210> 37
<211> 19
<212> DNA
<213> 人工序列
<400> 37
ctgaacgtca cctcacatg 19
<210> 38
<211> 19
<212> DNA
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atcaaccatc cgtgtcttc 19
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<211> 19
<212> DNA
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acctcaattt tcccctcta 19
<210> 40
<211> 20
<212> DNA
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atctggattt atgtctgctt 20
<210> 41
<211> 25
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cacatttgcc tcccactct 19
Claims (10)
1.一种sgRNA片段,其特征在于,所述sgRNA的DNA序列为5-’ACTCCACTCACTCCAGTCACCGG-3’。
2.一种基因打靶试剂盒,其特征在于,所述试剂盒包括权利要求1所述的sgRNA。
3.一种基因敲除方法,其特征在于,利用权利要求1所述的sgRNA片段识别目的基因上的双链上的不同靶位点,与一种核酸酶结合并通过识别靶位点处PAM序列,引导核酸酶结合到目的基因靶位点处,并开始进行随机剪切,随后从切口处脱落,形成DSB缺口,随后细胞通过非同源末端连接修复机制修复目的基因双链,造成移码突变,最终目的基因被敲除。
4.根据权利要求3所述基因敲除方法,其特征在于,所述sgRNA片段与核酸酶发挥作用的数量为一个或以上。
5.一种IL7R基因缺失斑马鱼突变体的制备方法,其特征在于,所述制备方法包括:
(1)将权利要求1所述的sgRNA和Cas9 mRNA共同导入斑马鱼中;
(2)培养获得稳定遗传的IL7R基因缺失斑马鱼突变体。
6.根据权利要求5所述的制备方法,其特征在于,所述sgRNA的获得步骤如下:
(1)确定IL7R基因敲除的靶位点;
(2)根据步骤(1)确定的靶位点序列设计扩增引物,PCR获得sgRNA的体外转录的模板;
(3)根据步骤(2)获得的模板进行体外转录。
7.根据权利要求6所述的制备方法,其特征在于,所述sgRNA的获得步骤如下:
(1)查询斑马鱼IL7R基因的基因组DNA序列及其功能结构域,根据CRISPR/Cas9敲除原理,设计一对IL7R基因的靶位点;
(2)PCR法获得sgRNA的体外转录的模板,正向引物序列为:5’TAATACGACTCACTATAGACTCCACTCACTCCAGTCACgttttagagctagaaatagc-3’,前18位碱基序列是T7启动子,19-38位碱基序列是sgRNA靶序列,小写字母表示的碱基序列为sgRNA上游骨架;反向引物序列为25bpsgRNA下游骨架,序列为5’-AAAAAAAGCACCGACTCGGTGCCAC-3’,按照下列PCR条件进行扩增:预变性95℃3min;变性95℃30s,退火54℃30s,延伸72℃20s进行35个循环,再72℃10min;PCR产物回收纯化;
(3)以RNA体外转录试剂盒进行体外转录。
8.根据权利要求5所述的制备方法,其特征在于,所述Cas9 mRNA是按照以下步骤获得的:
以T7驱动的人源化的Cas9编码序列为模板,用T7 RNA聚合酶进行Cas9体外转录,然后戴帽并加polyA尾;RNA经无水乙醇法沉淀,用DEPC处理过的超纯水重悬。
9.根据权利要求5所述的制备方法,其特征在于,sgRNA和Cas9 mRNA共同导入斑马鱼中,包括:将sgRNA和Cas9 mRNA混合,sgRNA终浓度为50ng/μL、Cas9 mRNA终浓度为250ng/μL,在斑马鱼胚胎1-4细胞期进行显微注射,注射量为1nL。
10.根据权利要求5所述的制备方法,其特征在于,培养获得稳定遗传的IL7R基因缺失斑马鱼突变体,包括:
(1)对经过注射的斑马鱼进行基因型鉴定,确定IL7R基因敲除的初建者F0;
(2)IL7R基因敲除的初建者F0个体进行自交获得F1;
(3)利用基因型鉴定的方法确定IL7R基因敲除的F1;
(4)从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代;
(5)鉴定为F2代中IL7R基因敲除的纯合子即为稳定遗传的IL7R基因缺失斑马鱼突变体。
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