CN111926017A - 一种csf1ra基因缺失斑马鱼突变体的制备及其应用 - Google Patents
一种csf1ra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csf1ra基因缺失斑马鱼突变体的制备及其应用,本发明利用CRISPR/Cas9技术设计csf1ra基因敲除sgRNA靶点,首次成功构建了csf1ra基因缺失斑马鱼突变体,此外,本发明的突变体生物模型可用于csf1ra基因功能机制的深入研究,包括csf1ra基因在神经***发育中的作用研究。
Description
技术领域
本发明涉及基因敲除技术领域,更具体地,涉及csf1ra基因缺失斑马鱼突变体的制备和应用。
背景技术
集落刺激因子受体(colony stimulating factor 1receptor,CSF1R)是III型受体型酪氨酸激酶血小板衍生生长因子(platelet-derived growth factor receptor,PDGFR)家族的一员,存在于人类、小鼠、大鼠、斑马鱼等物种中。生理情况下,CSF1R通过与配体IL-34的结合影响单核/巨噬细胞的增殖、分化及存活等行为。病理情况下,CSF1R的异常表达与多种疾病密切相关。在造血***中,CSF1R的突变可引发骨髓增生异常综合征和急性巨核细胞白血病等疾病;在非造血***中,CSF1R过表达可见于多种实体肿瘤,参与恶性肿瘤的增殖、转移和微环境调控等。因此,拓展对CSF1R功能的认识,对于深入理解其在相关疾病发生发展中的作用具有重要的意义。
成簇规律间隔短回文重复***(clustered regularly interspaced shortpalindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas),CRISPR/Cas)是细菌或古细菌在应对噬菌体长期选择压力下形成的一种获得性免疫防御机制,能够识别自身和外源入侵的DNA片段。CRISPR/Cas***主要分为3种类型:I型、II型和III型,其中II型CRISPR/Cas***含有标志性Cas9蛋白,即CRISPR/Cas9***。Cas9蛋白具有核酸内切酶活性,能够识别并切割DNA双链,当噬菌体入侵机体时,在向导RNA(single guide,sgRNA)指导下,Cas9蛋白对外源DNA进行切割形成双链DNA缺口,断裂的双链激活DNA损伤修复机制,再通过同源重组修复和非同源末端连接修复机制对断裂DNA进行修复,以实现靶基因的敲入和敲除。CRISPR/Cas9***具有作用高效、成本低廉、操作简单等优势,被广泛应用于精确和靶向性基因编辑。
在斑马鱼,CSF1R由csf1ra基因编码,在早期巨噬细胞即可表达。巨噬细胞首先在卵黄囊中分化,随后进入头部间质并迁移定植于大脑、视网膜等部位。在迁移过程中,进入头部的一部分巨噬细胞会转化为小胶质细胞,成为中枢神经***最主要的免疫细胞。因此,小胶质细胞由外周的造血组织分化而来,迁移至中枢神经***后在相对封闭的脑部环境中维持自我增殖与分化,维持神经***的稳态。本发明利用斑马鱼这一模型生物,通过CRISPR/Cas9基因编辑技术对其基因组进行编辑,实现目的基因的定位敲除,从而获得csf1ra基因敲除斑马鱼突变体,这将为后续的疾病建模和分子机制研究方面提供基础。
发明内容
基于此,本发明的目的在于提供一种csf1ra基因缺失斑马鱼突变体的制备及其应用,以斑马鱼为生物模型,通过CRISPR/Cas9基因编辑技术对其基因组进行编辑,实现目的基因的定位敲除,从而获得csf1ra基因敲除斑马鱼突变体。
本发明的目的是通过以下技术方案来实现的:
本发明的第一个方面提供了靶向斑马鱼csf1ra基因的sgRNA。
进一步,所述的sgRNA序列包括SEQ ID NO.1~5所示的核苷酸序列。
本发明还提供了一种csf1ra基因敲除方法,其特征在于,设计识别csf1ra基因靶位点的sgRNA序列,所述的sgRNA序列与核酸酶结合并引导核酸酶结合到csf1ra基因靶位点处,核酸酶对靶位点处的序列进行随机剪切,通过细胞自身的非同源末端连接修复机制修复csf1ra基因双链,造成移码突变,完成csf1ra基因被敲除。
进一步,所述的识别csf1ra基因靶位点的sgRNA序列包括SEQ ID NO.1~5所示的核苷酸系列,所述核酸酶为Cas9蛋白。
本发明的第二个方面提供了一种csf1ra基因缺失斑马鱼突变体的制备方法。
进一步,所述制备方法包括以下步骤:
(1)设计并合成识别csf1ra基因靶位点的sgRNA序列,所述的sgRNA序列包括SEQID NO.1~5所示的核苷酸系列;
(2)将所述的sgRNA序列和Cas9 mRNA共同注射到斑马鱼受精卵中,培养获得稳定遗传的csf1ra基因缺失斑马鱼突变体。
进一步,所述步骤(1)具体包括:查询斑马鱼csf1ra基因序列及功能结构域,结合CRISPR/Cas9敲除原理,设计并合成sgRNA序列,在sgRNA识别序列前添加T7启动子,后方添加sgRNA骨架,以此为模板,用T7 RNA聚合酶进行sgRNA体外转录。RNA经无水乙醇法沉淀,用DEPC处理过的超纯水重悬。
进一步,所述步骤(2)中的Cas9 mRNA是按照以下步骤获得的:以T7驱动的人源化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。
进一步,所述步骤(2)中的斑马鱼受精卵为1-4细胞期受精卵,显微注射剂量为1nL,其中sgRNA的注射浓度为50ng/μL,Cas9 mRNA的注射浓度为250ng/μL。
进一步,步骤(2)中所述培养获得稳定遗传的csf1ra基因缺失斑马鱼突变体包括以下步骤:
1)将经过注射的受精卵培养至成鱼,得到F0代csf1ra基因缺失的斑马鱼;
2)将得到的F0代csf1ra基因缺失的斑马鱼与野生型斑马鱼交配得到F1代斑马鱼,并鉴定F1代csf1ra基因缺失的斑马鱼;
3)将得到的F1代csf1ra基因缺失的雌性和雄性斑马鱼进行杂交得到F2代斑马鱼,并鉴定纯合的F2代csf1ra基因缺失的斑马鱼。
本发明的第三个方面提供了一种csf1ra基因缺失斑马鱼突变体的检测方法。
进一步,所述检测方法包括以下步骤:
以待检测的斑马鱼基因组DNA为模板,以根据目标序列设计的引物对进行PCR扩增,将扩增的PCR产物重组入pGEM-T载体后挑选单克隆,PCR结果验证后挑选阳性克隆测序,确认其等位基因的基因型,检测是否为csf1ra基因缺失斑马鱼突变体。
在本发明的具体实施方案中,F0和F1代csf1ra基因缺失斑马鱼突变体的检测中,根据目标序列设计的引物对为csf1ra-PF和csf1ra-PR(SEQ ID NO.6~7);挑选单克隆后,对转化子进行扩增时的引物对为T7和SP6,再辅以引物csf1ra-PF和csf1ra-PR进行PCR验证,验证后的克隆送检测序,测序引物为T7。F2代csf1ra基因缺失斑马鱼突变体的检测中,针对突变类型及位点设计的突变型引物对为csf1ra-mut-PF和csf1ra-mut-PR(SEQ IDNO.8~9),设计的野生型引物对为csf1ra-wt-PF和csf1ra-wt-PR(SEQ ID NO.10~11)。其中,按照下列PCR条件进行扩增:预变性95℃3min,变性95℃30s,退火56℃30s,延伸72℃50s,进行35个循环,72℃延伸10min。
本发明的第四个方面提供了csf1ra基因在制备神经***细胞缺损动物模型中的应用。
本发明的第五个方面提供了本发明第一个方面所述的sgRNA在构建csf1ra基因缺失斑马鱼突变体中的应用。
本发明的第六个方面提供了本发明第二个方面所述的制备方法制备的csf1ra基因缺失斑马鱼突变体在csf1ra基因研究功能中的应用。
本发明的csf1ra基因缺失斑马鱼突变体构建动机在于:在前期实验基础上,发现CSF1R参与神经***的发育,敲减其编码基因csf1ra可导致视网膜神经细胞发育延迟。因此有必要构建csf1ra基因缺失斑马鱼突变体,并以此为模型,对csf1ra基因对神经***发育的影响和CSF1R的功能进行在体实验研究。
本发明的优点和有益效果:
(1)本发明首次利用CRISPR/Cas9技术设计csf1ra基因敲除sgRNA靶点,实现斑马鱼中csf1ra基因的特异敲除。
(2)本发明基于csf1ra基因可能与神经***的发育有密切关系的前期实验结果,构建了国内外首例csf1ra基因缺失斑马鱼突变体。
(3)本发明构建的csf1ra基因缺失斑马鱼突变体可稳定遗传,便于后续对csf1ra基因功能机制的深入研究,包括csf1ra基因在神经***发育中的作用研究。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1显示csf1ra基因敲除sgRNA靶点设计示意图;
图2显示注射sgRNA2、4、6、7、8后的基因测序结果图;
图3显示csf1ra敲除单克隆筛选PCR检测结果图;
图4显示csf1ra敲除单克隆菌株序列与野生型序列比对结果图;
图5显示csf1ra敲除突变体蛋白质结构预测示意图;
图6显示F2代纯合子的鉴定及测序结果图,其中,A:电泳图、B:测序图、C:序列对比图;
图7显示斑马鱼纯合突变体csf1ra-/-检测脱靶效应的测试图;
图8显示Western Blot检测斑马鱼纯合突变体csf1ra-/-中csf1ra蛋白表达情况图。
具体实施方式
下面结合具体的实施例进一步说明本发明,本发明的实施例仅用于解释本发明,并不意味着限制本发明的保护范围。
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1sgRNA的筛选
1、实验动物
按照标准化方案养殖野生型斑马鱼(TU品系),养殖在自动循环养殖***中,水温为28.5℃,光照/黑暗周期为14h/10h,斑马鱼性成熟后进行交配,产卵后收集胚胎,养殖于1×Holt buffer(60mmol/L NaCl,0.67mmol/L KCl,0.3mmol/L NaHCO3,0.9mmol/L CaCl2,pH=7.2)孵化液中,以受精的小时数(hours post-fertilization,hpf)或受精的天数(dayspost-fertilization,dpf)表示胚胎或幼鱼的发育阶段。
2、CRISPR/Cas9基因敲除靶位点设计
在NCBI(https://www.ncbi.nlm.nih.gov/gene/)上查找斑马鱼csf1ra基因(Genbank NM_131672)的相关信息,获得该基因的蛋白质编码区(Coding sequence,CDS)区序列。利用在线网站MIT CRISPR online(http://crispr.mit.edu/)设计sgRNA的识别靶点。依据斑马鱼csf1ra基因的基因组DNA序列及其功能结构域,结合蛋白质保守功能结构域,将待敲除的靶点(sgRNA识别序列)设计在编码114aa之后的基因组序列上,即外显子(Exon)3和4,见图1。根据CRISPR/Cas9敲除原理,该靶点包含20个碱基,靶点的选择标准为:5’-GG-(N)18-NGG-3’,其中5’端的GG二核普酸是T7启动子的一部分,3’端是NGG(N为任意碱基)。
3、sgRNA活性检测
3.1实验步骤
针对外显子3和4共设计合成了8个sgRNA识别靶点,四个sgRNA一组,与Cas9 mRNA进行共注射。提取胚胎DNA选择直接测序法进行sgRNA活性检测。sgRNA序列表如表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2中提到的步骤。
表1 sgRNA序列
3.2实验结果
通过分析测序得到的图谱发现sgRNA1、sgRNA3、sgRNA5、sgRNA7混合组中sgRNA7有活性;sgRNA2、sgRNA4、sgRNA6、sgRNA8混合组中四个sgRNA均有活性,测序结果图见图2。因此,选择向受精卵中同时注射有活性的sgRNA2/4/6/7/8和Cas9 mRNA,以制备csf1ra基因缺失斑马鱼突变体。其中,sgRNA2/4/6/7/8对应的序列分别为:
sgRNA2:5’-GAGGAACGAGUAUGUAAAAC-3’(SEQ ID NO.1);
sgRNA4:5’-GACUUUUACUACAACGUCACA-3’(SEQ ID NO.2);
sgRNA6:5’-GGAUGCUCUCGAUGGCUAAA-3’(SEQ ID NO.3);
sgRNA7:5’-GUGUGCGACAGCUGAACAGA-3’(SEQ ID NO.4);
sgRNA8:5’-GUACGGGUCAGAACGAAGCU-3’(SEQ ID NO.5);
实施例2斑马鱼胚胎的显微注射及靶位点有效性测定
1、sgRNA的体外转录
在sgRNA识别序列前添加T7启动子部分(TAATACGACTCACTATA),后方添加sgRNA骨架。以此为模板,用T7 RNA聚合酶进行sgRNA体外转录。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。
2、Cas9 mRNA的体外转录
Cas9 mRNA以T7驱动的人源化Cas9编码序列为模板,用T7 RNA聚合酶进行体外转录(mMESSAGE mMACHINE T7,Thermo Fisher Scientific),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
收集斑马鱼胚胎,将sgRNA(终浓度为50ng/μL)和Cas9 mRNA(终浓度为250ng/μL)混合均匀,在斑马鱼胚胎1-4细胞期进行显微注射,每胚胎注射量为1nL。经注射的受精卵放置于1×Holt buffer(60mmol/L NaCl,0.67mmol/L KCl,0.3mmol/L NaHCO3,0.9mmol/LCaCl2,pH=7.2)孵化液中,28.5℃条件下孵化。
待胚胎发育至24hpf时,制备基因组DNA模板,以引物对csf1ra-PF和csf1ra-PR进行PCR扩增,反应条件为:预变性95℃3min,变性95℃30s,退火56℃30s,延伸72℃50s,进行35个循环,72℃延伸10min。具体的引物序列如下:
csf1ra-PF:5’-GTTTCCCTGTCCCTTCTGCT-3’(SEQ ID NO.6)
csf1ra-PR:5’-AGGGACCAACTACATTCACACA-3’(SEQ ID NO.7)
将扩增的PCR产物分别亚克隆到pGEM-T Easy载体中,以引物对T7和SP6对转化子进行扩增,再辅以引物csf1ra-PF和csf1ra-PR进行PCR电泳验证,最后依据条带大小,选取条带正确的克隆送测序,测序引物为T7,根据基因序列比对,检验是否出现突变以确定有活性的sgRNA。
实施例3 csf1ra基因敲除初建者的制备
按照实施例2所述的方法,在1-4细胞期向斑马鱼受精卵内同时注射有活性的sgRNA(50ng/μL)和Cas9 mRNA(250ng/μL),每胚胎注射量为1nL,按照实施例2中的方法对得到的csf1ra基因敲除的F0代进行鉴定,以确定csf1ra基因敲除的初建者F0(Founder,F0)。
实施例4 F1突变体筛选与鉴定
1、实验步骤
将初建者F0胚胎饲养至性成熟,与野生型(TU)杂交,获得F1胚胎。将F1胚胎按常规饲养至性成熟,取3~4月龄的F1成鱼,剪取部分尾鳍组织放入PCR管中,加入10μL YSYbuffer,经PCR反应(65℃30min,95℃5min,16℃1min,4℃)后制备基因组DNA模板,以引物对csf1ra-PF和csf1ra-PR进行基因组DNA片段PCR扩增。将目的基因csf1ra PCR产物重组入pGEM-T载体后挑选单克隆,PCR结果验证后挑选阳性克隆测序,确认其等位基因的基因型,以确认突变体携带的基因型。两个体系的反应条件均为预变性95℃3min,变性95℃30s,退火56℃30s,延伸72℃50s,进行35个循环,72℃延伸10min。
2、实验结果
PCR验证克隆电泳结果图见图3,体系一为T7/SP6扩增组,体系二为引物对csf1ra-PF/R扩增组。奇数泳道为验证体系一电泳结果,偶数泳道为验证体系二电泳结果,每相邻两个泳道为一个验证组,从图3可以看出该斑马鱼中3、12、14、16号为阳性克隆。
将阳性克隆PCR产物进行测序,测序结果显示F1突变体产生了-37bp+15bp-3bp的移码突变(见图4)。与野生型相比,移码突变造成了氨基酸序列的改变(见表2),由此产生截短蛋白(见图5)。以上结果表明敲除成功,出现了突变型斑马鱼。
表2基因组序列及预测的蛋白质编码序列表
实施例5F2代纯合突变体筛选与鉴定
1、实验步骤
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,30dpf后剪取部分尾鳍组织提取基因组DNA模板,进行PCR扩增并测序。针对突变类型及位点设计突变型引物对(csf1ra-mut-F/R)和野生型引物对(csf1ra-wt-F/R)。扩增及鉴定引物序列如下:
csf1ra-wt-PF(鉴定野生型):5’-GGGCGACCGTTTAGCCAT-3’(SEQ ID NO.8)
csf1ra-wt-PR(鉴定野生型):5’-TTGTGTTTAAACCCCTCAT-3’(SEQ ID NO.9)
csf1ra-mut-PF(鉴定突变体):5’-CAATGGAGGGCGACCATC-3’(SEQ ID NO.10)
csf1ra-wt-PR(鉴定突变体):5’-TTGTGTTTAAACCCCTCAT-3’(SEQ ID NO.11)
2、实验结果
由PCR验证电泳结果图可知1、2、3号鱼为野生型,4、5、6号鱼为纯合子,7、8、9号鱼为杂合子(见图6A)。通过PCR条带分析将电泳条带鉴定为纯合突变的鱼以csf1ra-mut-F1为引物进行测序来做进一步鉴定,测序峰图为单一条带(见图6B),序列对比图显示突变类型为-37bp+15bp-3bp(见图6C),可以确定为该斑马鱼为纯合突变体csf1ra-/-,可将此纯合子斑马鱼用于传代。
实施例6纯合突变体csf1ra-/-的脱靶效应的检测
1、实验方法
在http://crispr.mit.edu/网站上输入sgRNA2/4/6/7/8的序列,选取得分较高的6个潜在脱靶位点进行鉴定。所选脱靶位点如表3所示,针对脱靶位点所设计的引物如表4所示,通过PCR扩增相应的片段并测序,测序引物为off-target n F/R,通过测序序列和峰图判断是否出现脱靶效应。
2、实验结果
测序结果见图7,6个潜在脱靶位点处的突变体序列与野生型序列相一致,且无套峰出现,说明此csf1ra-/-纯合突变体不存在脱靶效应。
表3潜在脱靶位点的预测
表4针对预测脱靶基因所设计的引物序列
实施例7csf1ra基因缺失斑马鱼突变体在蛋白水平的鉴定
1、实验方法
蛋白印迹法。待csf1ra-/-纯合斑马鱼胚胎发育至48hpf,随机取野生型与csf1ra-/-突变体斑马鱼各30条,提取蛋白质并定量,进行蛋白印迹实验。所用抗体为:anti-CSF1R多克隆抗体(1:500,orb393256;Biorbyt,United Kingdom)。Anti-β-Actin单克隆抗体(1:3000,sc-47778;Santa Cruz Biotechnology Inc.)为内参照。
2、实验结果
以蛋白印迹方法检测斑马鱼纯合突变体csf1ra-/-中CSF1R蛋白的表达水平,结果见图8,与野生型相比,纯合突变体中无CSF1R的表达,表明通过CRISPR/Cas9技术成功构建了csf1ra缺失型的纯合斑马鱼突变体。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
<110> 南开大学
<120> 一种csf1ra基因缺失斑马鱼突变体的制备及其应用
<141> 2020-08-27
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Claims (10)
1.靶向斑马鱼csf1ra基因的sgRNA,所述的sgRNA的序列包括SEQ ID NO.1~5所示的核苷酸序列。
2.一种csf1ra基因缺失斑马鱼突变体的制备方法,其特征在于,所述的制备方法包括以下步骤:
1)设计并合成识别csf1ra基因靶位点的sgRNA序列,所述的sgRNA序列包括SEQ IDNO.1~5所示的核苷酸系列;
2)将所述的sgRNA序列和Cas9 mRNA共同注射到斑马鱼受精卵中,培养获得稳定遗传的csf1ra基因缺失斑马鱼突变体。
3.根据权利要求2所述的制备方法,其特征在于,所述步骤1)具体包括:查询斑马鱼csf1ra基因序列及功能结构域,结合CRISPR/Cas9敲除原理,设计并合成sgRNA序列,在sgRNA识别序列前添加T7启动子,后方添加sgRNA骨架,以此为模板,用T7 RNA聚合酶进行sgRNA体外转录。
4.根据权利要求2所述的制备方法,其特征在于,步骤2)中所述的Cas9mRNA是按照以下步骤获得的:以T7驱动的人源化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。
5.根据权利要求2所述的制备方法,其特征在于,步骤2)中所述的斑马鱼受精卵为1-4细胞期受精卵,显微注射剂量为1nL,其中sgRNA的注射浓度为50ng/μL,Cas9 mRNA的注射浓度为250ng/μL。
6.根据权利要求2所述的制备方法,其特征在于,培养获得稳定遗传的csf1ra基因缺失斑马鱼突变体包括以下步骤:
1)将经过注射的受精卵培养至成鱼,得到F0代csf1ra基因缺失的斑马鱼;
2)将得到的F0代csf1ra基因缺失的斑马鱼与野生型斑马鱼交配得到F1代斑马鱼,并鉴定F1代csf1ra基因缺失的斑马鱼;
3)将得到的F1代csf1ra基因缺失的雌性和雄性斑马鱼进行杂交得到F2代斑马鱼,并鉴定纯合的F2代csf1ra基因缺失的斑马鱼。
7.一种csf1ra基因缺失斑马鱼突变体的检测方法,其特征在于,包括以下步骤:
以待检测的斑马鱼基因组DNA为模板,以根据目标序列设计的引物对进行PCR扩增,将扩增的PCR产物重组入pGEM-T载体后挑选单克隆,PCR结果验证后挑选阳性克隆测序,确认其等位基因的基因型,检测是否为csf1ra基因缺失斑马鱼突变体。
8.csf1ra基因在制备神经***细胞缺损动物模型中的应用。
9.权利要求1所述的sgRNA在构建csf1ra基因缺失斑马鱼突变体中的应用。
10.根据权利要求2所述的制备方法制备的csf1ra基因缺失斑马鱼突变体在csf1ra基因功能研究中的应用。
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