CN114891791A - 特异性靶向犬Rosa26基因的sgRNA及其应用 - Google Patents
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Abstract
本发明提供特异性靶向犬Rosa26基因的sgRNA及其应用,所述sgRNA的靶向序列如SEQ ID NO:4、5、9、11所示。
Description
技术领域
本发明涉及生物技术领域,具体涉及特异性靶向犬Rosa26基因的sgRNA及其在犬基因组定点转基因技术中的应用。
背景技术
将外源基因或DNA片段***特定动物基因组中,观察和检测动物各种性状和表型的变化,既是基础科学研究基因功能的一个重要手段,也是经济动物改良品种品质的一个重要方法。早期的转基因生物一般采用随机***的方式将外源基因***基因组中,这种方法通常会产生以下问题:第一,由于外源基因***基因组的位置是随机的、不可预知的,因此有潜在的风险,造成动物基因组的***突变以及基因组的不稳定,对动物自身造成一些生理上的危害或导致一些非特异性的表型;第二,***基因的拷贝数不可控;第三,由于***位点的随机性,使得外源基因不能持续稳定表达,并且其遗传稳定性较差。因此,在技术允许的情况下,不管是在基础科学研究领域,还是在经济动物品种品质改良方面,将外源基因定点***基因组特定的“安全位点”都是一种更为安全、可靠和具有实用价值的技术。
CRISPR-Cas9是近年来发展极快的一种基因编辑技术,除了在基因敲除技术中的广泛应用,利用该技术还可大大提高基因敲入的概率。Cas9靶向切割DNA是通过一条向导RNA链,即sgRNA(single guide RNA)介导的。因此,sgRNA能否做到特异、精确靶向目标基因位点是CRISPR-Cas9能否行使其功能的先决条件。因此,设计、鉴定特异、精确靶向目标基因位点的sgRNA是CRISPR-Cas9技术的关键因素。
发明内容
Rosa26基因是一个在小鼠、猪、牛以及人类细胞等多个物种中常用的安全***位点。本发明在前期鉴定犬Rosa26基因位点的基础之上,设计、鉴定特异、精确靶向犬Rosa26基因的sgRNA,并阐述了这些sgRNA在Rosa26基因位点定点***外源基因中的应用。
具体来讲,本发明提供以下技术方案:
一方面,本发明提供特异性靶向犬Rosa26基因的sgRNA,其特征在于,所述sgRNA的靶向序列如SEQ ID NO:4、5、9、11所示。
另一方面,本发明提供试剂盒,其特征在于,所述试剂盒包括质粒,所述质粒包括如SEQ ID NO:4、5、9、11所示的序列。
另一方面,本发明提供表达载体,其特征在于,所述表达载体包括Cas9的序列和如SEQ ID NO:4、5、9、11所示的序列。
另一方面,本发明提供将外源基因定点***犬基因组的方法,其特征在于包括以下步骤:
(a)将如SEQ ID NO:4、5、9、11所示的序列连入含有sgRNA骨架序列的质粒中;
(b)将PCR扩增获得的外源基因序列、犬Rosa26基因同源重组的左臂核苷酸序列、犬Rosa26基因同源重组的右臂核苷酸序列与扩增载体连接,获得同源重组的供体载体;
(c)将步骤(a)所得的质粒、步骤(b)所得的供体载体和含有Cas9的质粒共同转染体细胞,所述体细胞选自犬成纤维细胞、皮肤细胞和肾小球细胞;
(d)采用PCR鉴定5’端同源重组结果和3’端同源重组结果,若PCR产物与预期相符,则表明已将外源基因定点***犬基因组。
在一些实施方案中,含有sgRNA骨架序列的质粒选自:addgene#51133质粒,addgene#42230质粒和addgene#58778质粒。
在一些实施方案中,所述犬Rosa26基因同源重组的左臂核苷酸序列如SEQ ID NO:12所示。
在一些实施方案中,所述犬Rosa26基因同源重组的右臂核苷酸序列如SEQ ID NO:13所示。
在一些实施方案中,所述扩增载体选自:addgene#61408质粒,addgene#22677质粒和addgene#51132质粒。
在一些实施方案中,含有Cas9的质粒选自:addgene#44758质粒,addgene#42230质粒和addgene#97307质粒。
另一方面,本发明提供供体载体,所述供体载体包含犬Rosa26基因同源重组的左臂核苷酸序列、犬Rosa26基因同源重组的右臂核苷酸序列。
另一方面,本发明提供菌株,所述菌株包含含有犬Rosa26基因同源重组的左臂核苷酸序列、犬Rosa26基因同源重组的右臂核苷酸序列的供体载体。
另一方面,本发明提供上述特异性靶向犬Rosa26基因的sgRNA在将外源基因定点***犬基因组中的应用。
另一方面,本发明提供上述特异性靶向犬Rosa26基因的sgRNA在将外源基因定点***犬基因组的转基因细胞模型的构建中的应用。
另一方面,本发明提供上述特异性靶向犬Rosa26基因的sgRNA在将外源基因定点***犬基因组转基因犬模型的构建中的应用。
另一方面,本发明提供上述特异性靶向犬Rosa26基因的sgRNA在外源基因在犬中过表达或功能研究中的应用。
定义
供体载体:供体载体在基因组同源重组过程中提供模板核苷酸序列,主要包含同源重组的左臂核苷酸序列,需要***的外源基因,以及同源重组的左臂核苷酸序列。
sgRNA:sgRNA(single guide RNA)也被称为向导RNA(guide RNA,gRNA),它是人工改造而成的可以在CRISPR基因编辑中引导Cas9蛋白在特定位点进行切割的一段RNA序列。它由骨架序列和一段与靶位点相配对的约20个碱基的特异序列组成。
同源重组:同源重组(Homologous Recombination)是指发生在非姐妹染色单体(non-sister chromatid)之间或同一染色体上含有同源序列的DNA分子之间或分子之内的重新组合。人们利用这一原理,可以实现外源基因在目的基因组内的定点整合或者定点敲除。
有益效果
本发明提供犬Rosa26基因的外源基因***位点,能够实现高效的基因编辑效率,不同sgRNA相应的编辑效率分别为sgRNA3:82.1%(32/39,39个克隆中有32个克隆是被编辑DNA序列),sgRNA4:76.5%(26/34),sgRNA8:92.9%(39/42),sgRNA10:84.2%(32/38)。
附图说明
图1示出不同sgRNA介导的Cas9切割效率电泳图。M1:MarkerIII,M2:D2000,1-10:sgRNA1-10。sgRNA介导的Cas9切割效率越高,大片段的DNA模板亮度就越低,被切割出的小片段DNA条带亮度就越高。从图中可以看出,sgRNA3、sgRNA4、sgRNA8、sgRNA10所介导的Cas9切割效率较高。
图2示出DOG-GFP-Rosa26质粒图谱。包含左臂、启动子、GFP、右臂等关键元件。
图3示出GFP基因定点***犬基因组Rosa26位点的示意图。图3A示出GFP基因定点***犬基因组Rosa26位点示意图,含有左臂、启动子pCMV、GFP和右臂的DNA序列与犬基因组发生同源重组,使得pCMV-GFP序列***到犬基因组Rosa26特定位点;图3B示出PCR鉴定5’端同源重组结果,图3C示出PCR鉴定3’端同源重组结果,其中左侧泳道为DNA分子量标准,中间泳道为PCR鉴定5’或3’端同源重组结果,右侧泳道为同源重组的阴性对照;图3D示出表达GFP的犬成纤维细胞,左侧图片为***pCMV-GFP序列的犬成纤维细胞,右侧为阴性对照。
图4示出pCMV-GFP序列同源重组到犬基因组后,5’同源重组PCR产物和3’同源重组PCR产物的Sanger测序鉴定结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,并参照附图,对本发明作进一步的详细说明。
实施例1设计、鉴定特异靶向犬Rosa26基因的sgRNA
犬Rosa26基因位于犬基因组第20条染色体,本发明所选择的sgRNA靶向序列位于犬Rosa26基因第一内含子,其序列如SEQ ID NO:1所示。分别以TAATACGACTCACTATAGGGGGAGTGCCGCAATACCTTTAGTTTTAGAGCTAGAAATAG(SEQ ID NO:2)、TAATACGACTCACTATAGGGGAGTTGCTCCTTCTCGATTGGTTTTAGAGCTAGAAATAG(SEQ ID NO:3)、TAATACGACTCACTATAGGGAGTTGCTCCTTCTCGATTGTGTTTTAGAGCTAGAAATAG(SEQ ID NO:4)、TAATACGACTCACTATAGGGGCTCCTTCTCGATTGTGGGCGTTTTAGAGCTAGAAATAG(SEQ ID NO:5)、TAATACGACTCACTATAGGGTGCTCCTTCTCGATTGTGGGGTTTTAGAGCTAGAAATAG(SEQ ID NO:6)、TAATACGACTCACTATAGGGTCAAACTGGGTAGCCTTAATGTTTTAGAGCTAGAAATAG(SEQ ID NO:7)、TAATACGACTCACTATAGGGATCAGTTCTACTGCCAATTAGTTTTAGAGCTAGAAATAG(SEQ ID NO:8)、TAATACGACTCACTATAGGGTGTCACAGCAACACGTTAGAGTTTTAGAGCTAGAAATAG(SEQ ID NO:9)、TAATACGACTCACTATAGGGGTGTATCACCTTCCCGTTGAGTTTTAGAGCTAGAAATAG(SEQ ID NO:10)、TAATACGACTCACTATAGGGGATGAATTCCATCAACGGGAGTTTTAGAGCTAGAAATAG(SEQ ID NO:11)为上游引物,以AAAAGCACCGACTCGGTGCC(SEQ ID NO:15)为下游引物,以包含有sgRNA骨架序列的质粒(购自addgene,编号为51133的质粒)为模板,进行PCR扩增,获得sgRNA体外转录模板,然后用体外转录试剂盒(购自NEB公司,货号E2040S)体外转录获得sgRNA。以TGGTACGGTCCTTCCCGAG(SEQ ID NO:16)为上游引物,以TTCCCACCCCTGTTTTAAGTGAA(SEQ ID NO:17)为下游引物,以比格犬基因组DNA为模板,进行PCR扩增并用胶回收试剂盒(购自ZYMORESEARCH公司,货号D4007)进行胶回收,回收得到的DNA可做为检测不同sgRNA和Cas9酶(购自Vazyme公司,货号#EN301)体外切割效率的模板DNA1(该模板中包含有与sgRNA特异靶向的DNA序列,可被sgRNA识别,从而介导Cas9对模板的切割反应)。按如下体积配制体外切割实验体系:
表1体外切割体系
无核酸酶水 | 补足至27ul |
Cas9核酸酶反应缓冲液 | 3ul |
sgRNA | 900nM |
Cas9核酸酶 | 1ul |
37℃孵育10分钟,加入3ul 30nM的模板DNA1,混匀后37℃孵育1小时,然后将反应产物进行琼脂糖凝胶电泳检测,结果如图1所示。最上端条带为模板DNA1琼脂糖凝胶电泳条带,该条带越弱,说明模板DNA1被切割越多,该sgRNA介导的Cas9体外切割效率越高。从中选取介导切割效率较高的sgRNA3、sgRNA4、sgRNA8、sgRNA10进行后续实验,同时以介导切割效率较低的sgRNA1为对照。
将sgRNA3(引物1:ACCGAGTTGCTCCTTCTCGATTGT,SEQ ID NO:18),引物2:AAACACAATCGAGAAGGAGCAACT,SEQ ID NO:19)、sgRNA4(引物1:ACCGGCTCCTTCTCGATTGTGGGC,SEQ ID NO:20,引物2:AAACGCCCACAATCGAGAAGGAGC,SEQ ID NO:21)、sgRNA8(引物1:ACCGTGTCACAGCAACACGTTAGA,SEQ ID NO:22,引物2:AAACTCTAACGTGTTGCTGTGACA,SEQ IDNO:23)、sgRNA10(引物1:ACCGGATGAATTCCATCAACGGGA,SEQ ID NO:24,引物2:AAACTCCCGTTGATGGAATTCATC,SEQ ID NO:25)靶向DNA序列连入含有sgRNA骨架序列的质粒(购自addgene,编号为51133的质粒),具体步骤如下:
1)限制性内切酶BsaI(购自NEB公司,货号R0535)酶切51133质粒后,用DNA纯化试剂盒(购自天漠生物,货号TD433)回收线性化的51133质粒DNA。
2)将100uM的引物1和引物2各5ul混合后按下列程序在PCR仪中进行退火配对:95℃,5分钟,85℃(60个循环,每个循环降1℃,15秒/循环),25℃(10个循环,每个循环降2℃,10秒/循环),4℃。
3)将退火配对后的引物与线性化的51133质粒DNA用T4 DNA连接酶(购自NEB公司,货号为M0202)进行连接。
4)将上述连接产物转化到Trans1-T1感受态细胞(购自全式金公司,货号CB111),第二天挑取单克隆菌落进行测序验证,选择正确连入51133质粒的单克隆菌株进行扩大培养,提取质粒DNA,得到可转录相应sgRNA的质粒51133-sgRNA3,51133-sgRNA4,51133-sgRNA8,51133-sgRNA10。
使用Amaxa Nucleo-fector II核转染仪和转染试剂(购自Lonza公司,货号VPI-1002),将4ug含有Cas9的质粒(购自addgene,编号48137)和2ug 51133-sgRNA3(或51133-sgRNA4或51133-sgRNA8或51133-sgRNA10)按照程序T-016转染到犬成纤维细胞,转染完成后用DMEM培养基(含20%血清,购自Invitrogen公司,货号C11995500BT),在5%CO2培养箱中培养48小时,换含有2ug/ml的嘌呤霉素的DMEM培养基继续培养,48h后收细胞进行PCR鉴定(PCR引物分别为P1F:CGCCTGAAGGACGAGACG,SEQ ID NO:26,P1R:ATCAGCCCGAGATGAATGCC,SEQ ID NO:27,P2F:GTCCGGTTGACCTTGACTGT,SEQ ID NO:28,P2R:TGCCATTCCAGAAGGAACCA,SEQ ID NO:29)。PCR反应体系和反应程序如表2和表3所示。
将4ul PCR产物和1ul pEASY-Blunt Cloning Vector(购自全式金公司,货号CB111)25℃孵育5分钟,然后将该连接产物转化到Trans1-T1感受态细胞(购自全式金公司,货号CB111),第二天挑取约40个单克隆菌落进行测序,计算不同sgRNA相应的编辑效率分别为sgRNA3(其靶向序列如SEQ ID NO:4所示):82.1%(32/39,39个克隆中有32个克隆是被编辑DNA序列),sgRNA4(其靶向序列如SEQ ID NO:5所示):76.5%(26/34),sgRNA8(其靶向序列如SEQ ID NO:9所示):92.9%(39/42),sgRNA10(其靶向序列如SEQ ID NO:11所示):84.2%(32/38),对照sgRNA1(其靶向序列如SEQ ID NO:2所示):13.5%(5/37,37个克隆中有5个克隆是被编辑DNA序列)。
实施例2在sgRNA对应Rosa26基因位点定点***外源基因
本实施例以在犬基因组Rosa26位点定点***GFP报告基因为例,阐述本发明所述sgRNA在定点***外源基因到犬Rosa26位点的应用,主要包括以下步骤:
(1)构建包含有犬Rosa26基因同源臂、启动子、GFP报告基因的质粒。以将GFP基因定点***sgRNA8靶向DNA位点为例:以addgene#61408质粒(购自addgene,编号为61408质粒)为模板,PacI和AscI双酶切(购自NEB公司,货号为R0547和R0558)获得质粒骨架序列;以
TCTGCCTCTGGAATCCTCTTCTGAGCCATCTTTAATTAACCGTTTACCGCCATGTTGGC(SEQ IDNO:30)和TGTTAGGACAGTTGTCACAGCAACACGTTggcgcgccATAGTCCTGTCGGGTTTCGCCA(SEQ IDNO:31)为引物扩增pmaxGFP质粒(Lonza)获得GFP序列;以比格犬肌肉组织基因组DNA为模板,ACGACTCACTATAGGGCGAATTGGAGCTCCCCTTAATGCCTGAAGGACGAGACGAGCTG(SEQ ID NO:32)和GCCAACATGGCGGTAAACGGTTAATTAAAGATGGCTCAGAAGAGGATTCCAGAGGCAGA(SEQ ID NO:33)为引物,进行PCR反应获得犬Rosa26基因同源重组的左臂DNA序列(DNA聚合酶购自Vazyme公司,货号P525),如SEQ ID NO:12所示;以TGGCGAAACCCGACAGGACTATggcgcgccAACGTGTTGCTGTGACAACTGTCCTAACA(SEQ ID NO:34)和GATATCAAGCTTATCGATACCGTCGACGccaaggaagagctccacagtggacaaatgca(SEQ ID NO:35)为引物,PCR获得犬Rosa26基因同源重组的右臂DNA序列,如SEQ ID NO:13所示;PCR反应体系和反应程序如下所示:
表2PCR反应体系
表3PCR反应程序
将以上所得质粒骨架序列、GFP序列、左臂序列、右臂序列,采用无缝连接的方式(购自Clone Smarter公司,货号C5891)获得同源重组的供体质粒DOG-GFP-Rosa26,具体操作如下:
表4无缝连接体系
质粒-骨架 | 60ng |
左臂(1kb) | 12ng |
GFP(2kb) | 24ng |
右臂(2kb) | 24ng |
2ⅹSeamless Master Mix | 5ul |
ddH<sub>2</sub>O | 补足至10ul |
50℃孵育15min,将产物转化到Trans1-T1感受态细胞(购自全式金公司,货号CB111),第二天挑取单克隆菌落进行测序验证,选择阳性的单克隆菌株进行扩大培养,提取质粒DNA。所获得DOG-GFP-Rosa26质粒序列如SEQ ID NO:14所示,质粒图谱如图2所示。
(2)将GFP序列定点***到犬基因组Rosa26 sgRNA8靶向DNA位点。
将6ug线性化的DOG-GFP-Rosa26质粒(SalI酶切DOG-GFP-Rosa26质粒后获得,SalI酶购自NEB公司,货号为R0138)、4ug Cas9质粒(购自addgene,编号48137)和2ug 51133-sgRNA8如上述转染方法共同转染犬成纤维细胞。转染完成后用DMEM培养基(含20%血清,购自Invitrogen公司,货号C11995500BT),在5%CO2培养箱中培养48小时,换含有2ug/ml嘌呤霉素的DMEM培养基继续培养,48h后收细胞进行PCR鉴定。PCR反应体系和反应程序如表2和表3所示。
用引物F1(TTCCCACCCCTGTTTTAAGTGAA,SEQ ID NO:36)和R1(CCCGTGAGTCAAACCGCTAT,SEQ ID NO:37)鉴定5’端同源重组结果,引物F2(GGCTACTACAGCTTCGTGGT,SEQ ID NO:38)和R2(ATCAATCCTCATGCTGGGCTC,SEQ ID NO:39)鉴定3’端同源重组结果(图3A)。F1/R1预期PCR产物大小为1800bp,F2/R2预期PCR产物大小为3211bp,如图3B-3C所示,F1/R1和F2/R2 PCR产物琼脂糖凝胶条带大小均符合预期。对F1/R1和F2/R2 PCR产物进行测序鉴定,测序结果如图4所示,表明GFP报告基因已按照预期目标准确***Rosa26sgRNA8靶向DNA位点。
为了进一步检测GFP报告基因在***位点的表达情况,将上述加入2ug/ml嘌呤霉素的细胞培养48小时后,每20-50个细胞接种至10cm培养皿中,隔天换新鲜培养基;待单克隆形成后,用克隆环将细胞克隆点挑选出来,接种至48孔细胞培养板中进行扩繁;待细胞生长至80%汇合后,用胰酶消化收集细胞。每孔细胞分为两部分,一部分进行PCR实验,鉴定GFP基因***情况;PCR鉴定阳性的单克隆细胞,用共聚焦显微镜采集图片,鉴定GFP基因表达情况,如图3D所示细胞表达GFP荧光,表明***到犬基因组的GFP基因已稳定表达。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.特异性靶向犬Rosa26基因的sgRNA,其特征在于,所述sgRNA的靶向序列如SEQ IDNO:4、5、9、11所示。
2.试剂盒,其特征在于,所述试剂盒包括质粒,所述质粒包括如SEQ ID NO:4、5、9、11所示的序列。
3.表达载体,其特征在于,所述表达载体包括Cas9的序列和如SEQ ID NO:4、5、9、11所示的序列。
4.将外源基因定点***犬基因组的方法,其特征在于包括以下步骤:
(a)将如SEQ ID NO:4、5、9、11所示的序列连入含有sgRNA骨架序列的质粒中;
(b)将PCR扩增获得的外源基因序列、犬Rosa26基因同源重组的左臂核苷酸序列、犬Rosa26基因同源重组的右臂核苷酸序列与扩增载体连接,获得同源重组的供体载体;
(c)将步骤(a)所得的质粒、步骤(b)所得的供体载体和含有Cas9的质粒共同转染体细胞,所述体细胞选自犬成纤维细胞、皮肤细胞和肾小球细胞;
(d)采用PCR鉴定5’端同源重组结果和3’端同源重组结果,若PCR产物与预期相符,则表明已将外源基因定点***犬基因组。
5.根据权利要求4所述的将外源基因定点***犬基因组的方法,其特征在于,含有sgRNA骨架序列的质粒选自:addgene#51133质粒,addgene#42230质粒和addgene#58778质粒。
6.根据权利要求4或5所述的将外源基因定点***犬基因组的方法,其特征在于,所述犬Rosa26基因同源重组的左臂核苷酸序列如SEQ ID NO:12所示。
7.根据权利要求4-6中任一项所述的将外源基因定点***犬基因组的方法,其特征在于,所述犬Rosa26基因同源重组的右臂核苷酸序列如SEQ ID NO:13所示。
8.根据权利要求4-7中任一项所述的将外源基因定点***犬基因组的方法,其特征在于,所述扩增载体选自:addgene#61408质粒,addgene#22677质粒和addgene#51132质粒。
9.根据权利要求4-8中任一项所述的将外源基因定点***犬基因组的方法,其特征在于,含有Cas9的质粒选自:addgene#44758质粒,addgene#42230质粒和addgene#97307质粒。
10.如SEQ ID NO:4、5、9、11所示的序列在将外源基因定点***犬基因组中、将外源基因定点***犬基因组的转基因细胞模型的构建中、将外源基因定点***犬基因组转基因犬模型的构建中或在外源基因在犬中过表达或功能研究中的应用。
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