CN113862266A - 靶向小鼠BBS5基因的gRNA及构建Bardet–Biedl小鼠模型的方法 - Google Patents
靶向小鼠BBS5基因的gRNA及构建Bardet–Biedl小鼠模型的方法 Download PDFInfo
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Abstract
本发明属分子生物学领域,具体公开了一组靶向小鼠BBS5基因的gRNA及构建Bardet–Biedl小鼠模型的方法;本发明设计了特异性靶向BBS5基因的gRNA,利用cas9将BBS5基因的第3个外显子到第7个外显子敲除,造成移码突变,从而达到将该基因敲除的目的。该方法简单易行,周期短,在小鼠受精卵内就可实施,获得阳性克隆的几率很高。由该方法获得的BBS5基因敲除小鼠是作为研究BBS综合征的最佳模型,利用该小鼠模型可以充分研究该疾病的致病机理,并进一步开发针对该疾病的治疗方式。
Description
技术领域
本发明涉及生物技术领域,具体公开了能够有效敲除小鼠BBS5基因的gRNA 序列及其应用。
背景技术
巴迪特-比德尔综合征(Bardet–Biedl Syndrome,BBS)是一种遗传性疾病,影响身体的许多部位。患有这种综合症的人由于视锥、视杆营养不良导致的渐进性视力障碍;额外的手指或脚趾;肥胖症;男性性腺功能下降:肾脏异常;和学习困难等异常表型。通常开始于儿童发育期,早期存在夜盲问题,然后是周围视力盲点的发展,最终发展成隧道视觉。大多数人还会导致***或成年早期失明,身体异常包括多指(后轴多面体)肾脏问题(多囊肾)肥胖症,以及额外的生殖器异常和***不育,学习障碍等。目前已经知道这种疾病是隐性遗传的,许多基因的突变会导致巴迪特-比德尔综合征。目前已经发现有12个BBS基因,针对该疾病无有效的治疗方式。BBS5基因编码了与BBS综合征相关的蛋白质,细胞内的实验表明,这种基因在细胞中表达,是形成纤毛结构所需的蛋白。BBSome复合物被认为是将特定囊泡膜蛋白分拣到纤毛膜发育所需。Rab8 (GTP)进入原生纤毛,促进纤毛膜的延伸。实验动物疾病模型对于研究人类疾病发生的病因、发病机制、开发防治技术和开发药物是不可缺少的研究工具。为了研究该疾病的发病机理以及开发有效治疗方式,构建该疾病的小鼠模型对于该疾病的研究不可或缺,目前还缺乏有效的构建Bardet–Biedl小鼠模型的方法。
发明内容
针对现有技术不足,本发明设计了简单有效的方式,能利用Crispr/Cas9***快速在小鼠体内将BBS5基因敲除,构建该疾病的模型。 Crispr/Cas9技术能够在gRNA 的指引下在精准切割DNA造成双链断裂,在DNA双链修复时将突变引入基因组DNA中(见附图1)。同时在两个gRNA的指引下,基因组中的大片段DNA能够被切除。Cas/sgRNA复合物行使功能需要PAM序列,不同Cas酶,其对应的PAM并不完全相同。gRNA通常包括:靶标结合区和Cas蛋白识别区。靶标结合区与Cas蛋白识别区通常以5’到3’的方向连接。靶标结合区的长度通常为15~25个碱基,更通常为18~22个碱基,如20个碱基。靶标结合区与DNA的模板链特异性结合,从而将Cas9招募到预定位点。通常,DNA模板链上gRNA结合区域的对侧区紧邻PAM,或者隔开数个碱基(例如10个以内,或8个以内,或5个以内)。
本发明包括以下技术方案:
一组靶向小鼠BBS5基因的gRNA,包括两条gRNA,其核酸序列分别为:
gRNA1=SEQ ID NO 1=5’- ACTCTTACTATCCTATCCACTGG-3’;
gRNA2= SEQ ID NO 2= 5’ - TTCAATCCCCATGACGCTCATGG-3’。
进一步的,一种靶向小鼠BBS5基因的基因敲除试剂盒,所述试剂盒中包括如上述一组gRNA。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述方法包括利用gRNA1和gRNA2将小鼠BBS5基因敲除。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述gRNA1和gRNA2分别靶向BBS5基因第3个外显子的5'端和第7个外显子的3'端。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述gRNA1和gRNA2将BBS5基因第3~第7个外显子之间的6092bp的DNA切除,切除后的基因在转录形成的mRNA第2个外显子与第8个外显子剪切时融合,翻译蛋白质时形成移码突变。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,包括以下步骤:
1)将上述两条gRNA与Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0小鼠;
2)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;
3)待雄性Founder小鼠到8周龄,雌性小鼠到6周龄,分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示基因敲除成功。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述步骤1中的测序引物为:
PCR测序引物=SEQ ID NO 3=5’-GCCTTCATTGCTCTAGTGTTAGGA-3。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述步骤3中PCR鉴定中的引物包括:
PCR Primers1,其序列分别为:
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R1= SEQ ID NO 5=5’-GCCTTCATTGCTCTAGTGTTAGGA-3’。
进一步的,使用上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,所述步骤3中PCR鉴定中的引物包括:
PCR Primers2,其序列分别为:
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R2=SEQ ID NO 6=5’-ACAAGAGCCAGAGGTCCTGGG-3’。
进一步的,由上述靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法获得的Bardet–Biedl 综合症模型小鼠。
本发明具有以下有益效果:
BBS5是真核生物中最保守的蛋白,包含一个PH结构域和一个三螺旋结构,本发明敲除第3到第7个外显子是BBS5基因中的最重要的功能域,同时该方法不会引入外源序列。本发明设计的gRNA通过验证能够在小鼠受精卵中将BBS5基因的第3到第7个外显子高效且快速敲除,测序结果证实了小鼠体内BBS5基因的敲除。其中,所述的gRNA序列在待改变的BBS5基因上的靶序列上是唯一的。
该方法简单易行,周期短,在小鼠受精卵内就可实施,获得阳性克隆的几率很高。由该方法获得的BBS5基因敲除小鼠是作为研究BBS综合征的最佳模型,利用该小鼠模型可以充分研究该疾病的致病机理,并进一步开发针对该疾病的治疗方式。
附图说明
附图1 BBS5 基因敲除小鼠的构建方案示意图;图中,深色线段表示敲除区域,淡色矩形代表BBS5基因的外显子,gRNA region代表gRNA的结合区域。 F1, R1 代表用于小鼠鉴定的引物结合区域;
附图2 BBS5基因敲除小鼠的鉴定策略;显示了用于鉴定小鼠的PCR 引物(Annealing Temperature 60.0 ºC)的结合位点:F1、R1、R2;
附图3 BBS5基因敲除小鼠的鉴定结果;其中左图为DNA分子量标记,右图为PCR的鉴定产物图;基因敲除后BBS5等位基因的扩增产物大小582 bp ;野生型BBS5等位基因的扩增产物: 6673 bp;使用的阴性对照为:water:不加DNA模板;WT:400 ng小鼠基因组DNA;
附图4 BBS5基因敲除小鼠的基因组测序结果图;箭头指示处表明BBS5基因被删除了6092个碱基。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。本发明中所述的适量,为本领域内普通技术人员根据国家技术规范和生产实际情况所决定的用量。本发明中所述的原料如无特殊说明,均为商业购得。
实施例
1.两条gRNA的序列设计
根据小鼠BBS5基因的序列,设计并合成了2条针对BBS5基因的gRNA的序列:
gRNA1=SEQ ID NO 1=5’- ACTCTTACTATCCTATCCACTGG-3’;
gRNA2= SEQ ID NO 2= 5’ -TTCAATCCCCATGACGCTCATGG-3’
2. BBS5基因敲除小鼠的获得
Cas9/sgRNA的显微注射:4周龄的雌鼠先注射30单位的PMSG(血清***,Sigma),48小时后注射30单位的hCG(Sigma),随即将雌鼠与雄鼠交配。次日,获得小鼠受精卵。受精卵在KSOM(Millipore)中37℃,5%CO 2 培养2小时。将含有Cas9mRNA(25ng/μl),gRNA1和gRNA2 (各10ng/μl)的混合液通过显微注射至小鼠受精卵胞质。将注射后的受精卵移植到假孕母鼠输卵管内,经过20天孕育生产后,最终可获得F0小鼠,将F0代小鼠利用PCR技术进行检验,验证基因组中的BBS5基因被敲除,获得BBS5基因敲除阳性小鼠;待雄性F0小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,将筛选的阳性小鼠通过杂交和自交的方式,扩大种群数量,建立稳定的BBS5基因敲除小鼠.
3. BBS5基因敲除小鼠的PCR鉴定
根据BBS5基因的被敲除区域,在第3个外显子5’端以及第7个外显子3’端设计一对引物,F1和R1,鉴定策略图见附图2。
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R1= SEQ ID NO 5=5’-GCCTTCATTGCTCTAGTGTTAGGA-3’。
此对引物在基因敲除小鼠中扩增产物为582bp,在野生型小鼠中不能扩增出产物。
另外,在在第3个外显子5’端以及被敲除区域上设计一对引物F1和R2,
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R2=SEQ ID NO 6=5’-ACAAGAGCCAGAGGTCCTGGG-3’。
此对引物在野生小鼠中扩增产物为665bp,在基因敲除小鼠中不能扩增出产物。
基因组DNA提取:小鼠PCR鉴定部位为鼠尾,对于鼠尾鉴定选择裂解的方法提取DNA。
PCR鉴定反应体系如下:
小鼠基因组 DNA 2 μl
F1引物 (10 μM) 2 μl
R1引物 (10 μM) 2 μl
dNTPs (2.5 mM) 6 μl
5X LongAmp Taq 反应体系10 μl
LongAmp Taq DNA 聚合酶 2 μl
ddH 2 O 26 μl
总共 50 μl
反应程序:
预变性 94C 10min
变性 94C 20S
退火 58C 25S
延伸 72C 20S
终延伸 72C 5min
将PCR产物进行琼脂糖凝胶电泳,获得结果如图3所示,显示多数小鼠为阳性,本发明基因敲除的成功率高。
4 BBS5 基因敲除小鼠的基因组测序
将小鼠基因组DNA提取后使用引物F1R1进行扩增,将扩增产物送往苏州唯智公司进行测序,测序结果如附图4所示,显示成功获得BBS5基因敲除的小鼠。
通过以上实验1-4可知,本发明设计了特异性靶向BBS5基因的gRNA,利用cas9将BBS5基因的第3个外显子到第7个外显子敲除,造成移码突变,从而达到将该基因敲除的目的。该方法简单易行,周期短,在小鼠受精卵内就可实施,获得阳性克隆的几率很高。
以上仅为本发明的较佳实施例而已,不能以此限定本发明的保护范围,即大凡依本发明权利要求书及发明内容所做的简单的等效变化与修改,皆仍属于本发明专利申请的保护范围。
SEQUENCE LISTING
<110> 赛业(苏州)生物科技有限公司
<120> 靶向小鼠BBS5基因的gRNA及构建Bardet-Biedl小鼠模型的方法
<130> 2021
<160> 6
<170> PatentIn version 3.5
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Claims (10)
1.一组靶向小鼠BBS5基因的gRNA,其特征在于,包括两条gRNA,其核酸序列分别为:
gRNA1=SEQ ID NO 1=5’- ACTCTTACTATCCTATCCACTGG-3’;
gRNA2= SEQ ID NO 2= 5’ -TTCAATCCCCATGACGCTCATGG-3’。
2.一种靶向小鼠BBS5基因的基因敲除试剂盒,其特征在于,所述试剂盒中包括如权利要求1中的一组靶向小鼠BBS5基因的gRNA。
3.使用如权利要求1所述的一组靶向小鼠BBS5基因的gRNA构建Bardet–Biedl 综合症小鼠模型的方法,其特征在于,所述方法包括利用gRNA1和gRNA2将小鼠BBS5基因敲除。
4.根据权利要求3所述的方法,其特征在于,所述gRNA1和gRNA2分别靶向BBS5基因第3个外显子的5'端和第7个外显子的3'端。
5.根据权利要求4所述的方法,其特征在于,所述gRNA1和gRNA2将BBS5基因第3~第7个外显子之间的6092bp的DNA切除,切除后的基因在转录形成的mRNA第2个外显子与第8个外显子剪切时融合,翻译蛋白质时形成移码突变。
6.根据权利要求3所述的方法,其特征在于,包括以下步骤:
1)将上述两条gRNA与Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0小鼠;
2)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;3)待雄性Founder小鼠到8周龄,雌性小鼠到6周龄,分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示基因敲除成功。
7.根据权利要求6所述的方法,其特征在于,所述步骤1中的测序引物为:
PCR测序引物=SEQ ID NO 3=5’-GCCTTCATTGCTCTAGTGTTAGGA-3。
8.根据权利要求6所述的方法,其特征在于,所述步骤3中PCR鉴定中的引物包括:
PCR Primers1,其序列分别为:
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R1= SEQ ID NO 5=5’-GCCTTCATTGCTCTAGTGTTAGGA-3’。
9.根据权利要求6所述的方法,其特征在于,所述步骤3中PCR鉴定中的引物包括:
PCR Primers2,其序列分别为:
F1=SEQ ID NO 4=5’-AGAGCCAGATACGGTAGTGCATGCT-3’
R2=SEQ ID NO 6=5’-ACAAGAGCCAGAGGTCCTGGG-3’。
10.如权利要求3-8任一项所述的方法构建获得的Bardet–Biedl 综合症模型小鼠。
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