CN106957858A - 一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法 - Google Patents

一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法 Download PDF

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CN106957858A
CN106957858A CN201610854587.1A CN201610854587A CN106957858A CN 106957858 A CN106957858 A CN 106957858A CN 201610854587 A CN201610854587 A CN 201610854587A CN 106957858 A CN106957858 A CN 106957858A
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陈玉林
王小龙
蔡蓓
牛怡源
马宝华
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Abstract

本发明提供了一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法。本发明首先获得针对绵羊MSTN第二外显子和第三外显子的sgRNA识别区的两段DNA序列、针对绵羊ASIP第五外显子的sgRNA识别区的两段DNA序列、针对绵羊BCO2第二外显子的sgRNA识别区的两段DNA序列，分别设计并和成相应的sgRNA寡核苷酸序列；接着分别构建针对绵羊MSTN第二外显子和第三外显子、针对绵羊ASIP第五外显子、针对绵羊BCO2第二外显子，且含T7启动子的saRNA体外转录载体；利用Cas9和sgRNA的体外转录载体通过体外转录获得Cas9 mRNA和sgRNA，通过受精卵注射可用于生产MSTN、ASIP、BCO2共同敲除的转基因绵羊。

Description

一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基 因的方法
技术领域
本发明涉及动物基因工程和遗传修饰领域，具体涉及一种利用 CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法。
背景技术
CRISPR/Cas系统是细菌和古细菌内通过RNA介导的特异性切外源遗传物质的获得性免疫系统。II型CRISPR/Cas系统即CRISPR/Cas9已经被证明可以在试管中高效切割任意给定的DNA。CRISPR/Cas9与传统的ZFN和TALEN 技术相比效率高、序列选择限制小(只需要基因组上出现GG即可)，并且其构建过程简单，针对每个基因只需构建合适的sgRNA。但CRISPR/Cas9在哺乳动物细胞中会引起严重的脱靶效应，利用Cas9切口酶加上两个相背着的 PAM、距离比较近并且可以结合在不同链上的sgRNA可以大大降低脱靶效率。
传统的育种方法存在着育种年限长、一次选择性状数量有限等缺点，分子标记辅助选择仍处于理论发展迅速但是很难应用于生产实践的问题，转基因育种技术与传统育种技术的结合显得尤为重要。
与生长相关的重要基因——MSTN，即肌生成抑制素，又称生长/分化因子-8(GDF-8)。哺乳动物中MSTN基因主要在骨骼肌中表达，该基因在非翻译区的变异位点(A＞G)产生了干扰的靶位点，抑制了MSTN基因的翻译过程和调控功能，从而影响了动物肌肉的发育。
ASIP即刺鼠信号蛋白基因被证明在黑色素合成信号通路中其重要作用， ASIP基因高表达与绵羊被毛呈白色显著相关。ASIP在皮肤中表达的ASIP蛋白通过与α-MSH(黑色素细胞刺激激素)竞争性结合MC1R(黑色素皮质激素受体1)，使MC1R结构改变，抑制环磷酸腺苷酶系统引起cAMP水平下降，通过级联反应抑制真黑色素的形成，产生褐黑色素。过高表达的ASIP基因有可能还会造成黑色素合成减少从而使被毛颜色表现为白色。绵羊的农业价值不仅在于提供羊肉，羊毛和羊皮也是绵羊重要的产品，但是绵阳毛色的不整齐严重影响毛皮品质及后期的染色加工处理。
通过CRISPR/Cas9系统对绵羊基因组中抑制肌肉生长的MSTN基因、影响毛皮品质的ASIP基因和带来黄脂疾病的BCO2基因通过CRISPR/Cas9进行共同敲除，生产出肌肉发达、毛皮品质高、活力好的绵羊对我国畜牧业的长足发展具有重要意义。
发明内容
本发明提供了一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、 BCO2基因的方法。
为实现上述目的，本发明采取的技术方案为：一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其特征在于，包括以下步骤：
(1)构建特异性靶向MSTN第二外显子和第三外显子、ASIP第五外显子、BCO2第二外显子的sgRNA的体外转录载体；通过体外转录得到针对 MSTN第二外显子和第三外显子的sgRNA-1M、sgRNA-2M，针对ASIP第五外显子的sgRNA-1A、sgRNA-2A，针对绵羊BCO2第二外显子的sgRNA-1B、 sgRNA-2B；
(2)体外转录Cas9蛋白的体外转录载体，得到Cas9mRNA；
(3)将步骤(1)和步骤(2)的sgRNA-1M、sgRNA-2M、sgRNA-1A、 sgRNA-2A、sgRNA-1B、sgRNA-2B及Cas9mRNA纯化后测浓度，混合，注射入绵羊受精卵细胞质中，然后经体外培养后移植入同种雌性绵羊输卵管中，用于生产共同敲除MSTN、ASIP、BCO2基因的转基因绵羊。
本发明所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、 BCO2基因的方法，其特征在于，sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B与Cas9mRNA混合后，终浓度为Cas9 mRNA 20ng/μL、sgRNA-1M 5ng/μL、sgRNA-2M 5ng/μL、sgRNA-1A 5ng/μL、 sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
本发明所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、 BCO2基因的方法，其特征在于，所述sgRNA-1M、sgRNA-2M、sgRNA-1A、 sgRNA-2A、sgRNA-1B、sgRNA-2B的表达载体为pUC57-T7-gRNA，Cas9蛋白的体外转录载体为pST1374-NLS-flag-linker-Cas9。
本发明所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、 BCO2基因的方法，其特征在于，所述特异性靶向为绵羊MSTN第二外显子和第三外显子的sgRNA-1M、sgRNA-2M，针对绵羊ASIP第五外显子的 sgRNA-1A、sgRNA-2A，针对绵羊BCO2第二外显子的sgRNA-1B、sgRNA-2B。
本发明还提供了上述利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、 BCO2基因的方法的应用。
(1)将sgRNA-1M、sgRNA-2M共同或单独与Cas9mRNA同时转染至细胞，用于研究MSTN基因的功能；
(2)将sgRNA-1M、sgRNA-2M共同或单独与Cas9mRNA同时注射入受精卵，然后通过胚胎移植用于生产靶向敲除MSTN基因的转基因绵羊；
(3)将sgRNA-1M、sgRNA-2M共同或单独与Cas9mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞通过核移植的方法生产靶向敲除MSTN 基因的转基因绵羊；
(4)将sgRNA-1A、sgRNA-2A共同或单独与Cas9mRNA同时转染至细胞，用于研究ASIP基因的功能；
(5)将sgRNA-1A、sgRNA-2A共同或单独与Cas9mRNA同时注射入受精卵，然后通过胚胎移植用于生产靶向敲除ASIP基因的转基因绵羊；
(6)将sgRNA-1A、sgRNA-2A共同或单独与Cas9mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞通过核移植的方法生产靶向敲除ASIP基因的转基因绵羊。
(7)将sgRNA-1B、sgRNA-2B共同或单独与Cas9mRNA同时转染至细胞，用于研究BCO2基因的功能；
(8)将sgRNA-1B、sgRNA-2B共同或单独与Cas9mRNA同时注射入受精卵，然后通过胚胎移植用于生产靶向敲除BCO2基因的转基因绵羊；
(9)将sgRNA-1B、sgRNA-2B共同或单独与Cas9mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞通过核移植的方法生产靶向敲除BCO2 基因的转基因绵羊；
(10)将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B与Cas9mRNA同时注射入受精卵，然后通过胚胎移植用于生产共同敲除MSTN、ASIP、BCO2基因的转基因绵羊；
(11)将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B与Cas9mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞通过胚胎移植用于生产共同敲除MSTN、ASIP、BCO2基因的转基因绵羊；
(12)将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B中任意两个或两个以上sgRNA与Cas9mRNA同时注射入受精卵，然后通过胚胎移植用于生产特异靶向敲除的转基因绵羊；
(13)将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B中任意两个或两个以上sgRNA与Cas9mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞通过胚胎移植用于生产特异靶向敲除的转基因绵羊。
CRISPR/Cas9系统的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA，可以改造形成具有引导作用的sgRNA (singleguide RNA)，足以引导Cas9对DNA的定点切割。作为一种RNA导向的dsDNA结合蛋白，Cas9效应物核酸酶是已知的第一个统一因子(unifyingfactor)，能够共定位RNA、DNA和蛋白，从而拥有巨大的改造潜力。将蛋白与无核酸酶的Cas9(Cas9nuclease-null)融合，并表达适当的sgRNA，可靶定任何dsDNA序列，而sgRNA的末端可连接到目标DNA，不影响Cas9 的结合。因此，Cas9能在任何dsDNA序列处带来任何融合蛋白及RNA，这为生物体的研究和改造带来巨大潜力。
本发明的基因序列：
1、利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其所述MSTN、ASIP、BCO2基因特异性靶位点序列见表1。
表1
2、利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其所述MSTN、ASIP、BCO2基因设计的sgRNA序列见表2。
MSTN sgRNA-1top strand tagGTCTCAGATATATCCACAGT
MSTN sgRNA-1bottom strand aaacACTGTGGATATATCTGAGA
MSTN sgRNA-2top strand TAGGATTTTGAAGCTTTTGGAT
MSTN sgRNA-2bottom strand aaacATCCAAAAGCTTCAAAAT
ASIP sgRNA-1top strand ccggCTTCAGGTTCCTTTCATCTC
ASIP sgRNA-1bottom strand aaacGAGATGAAAGGAACCTGAAG
ASIP sgRNA-2top strand CCGGCAATTCTTCCATGAACCTGT
ASIP sgRNA-2bottom strand aaacACAGGTTCATGGAAGAATTG
BCO2sgRNA-1top strand ccgGTTAGAAGCGGTGCAATGCA
BCO2sgRNA-1bottom strand aaacTGCATTGCACCGCTTCTAA
BCO2sgRNA-2top strand ccGGTCGTCTCAGCTCGAGTCC
BCO2sgRNA-2bottom strand aaacGGACTCGAGCTGAGACGA
表2
3、利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其敲除MSTN、ASIP、BCO2基因的绵羊生产出的羔羊检测时所用引物序列见表3。
表3
具体实施方式
为了使本发明的目的及优点更加清楚明白，以下结合实施例对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例提供了一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、 ASIP、BCO2基因的方法，包括以下步骤：
(1)构建特异性靶向MSTN第二外显子和第三外显子、ASIP第五外显子、BCO2第二外显子的sgRNA的体外转录载体；通过体外转录得到针对MSTN第二外显子和第三外显子的sgRNA-1M、sgRNA-2M，针对ASIP第五外显子的sgRNA-1A、sgRNA-2A，针对绵羊BCO2第二外显子的sgRNA-1B、 sgRNA-2B；
(2)体外转录Cas9蛋白的体外转录载体，得到Cas9mRNA；
(3)将步骤(1)和步骤(2)的sgRNA-1M、sgRNA-2M、sgRNA-1A、 sgRNA-2A、sgRNA-1B、sgRNA-2B及Cas9mRNA纯化后测浓度，混合，注射入绵羊受精卵细胞质中，然后经体外培养后移植入同种雌性绵羊输卵管中，用于生产共同敲除MSTN、ASIP、BCO2基因的转基因绵羊。
其中sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B与Cas9mRNA混合后，终浓度为Cas9mRNA 20ng/μL、sgRNA-1M 5ng/μL、sgRNA-2M 5ng/μL、sgRNA-1A 5ng/μL、sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
所述sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、 sgRNA-2B的表达载体为pUC57-T7-gRNA，Cas9蛋白的体外转录载体为 pST1374-NLS-flag-linker-Cas9。
所述特异性靶向为绵羊MSTN第二外显子和第三外显子的sgRNA-1M、 sgRNA-2M，针对绵羊ASIP第五外显子的sgRNA-1A、sgRNA-2A，针对绵羊 BCO2第二外显子的sgRNA-1B、sgRNA-2B。
步骤一：针对MSTN、ASIP、BCO2基因的CRISPR/Cas9系统的构建
1、根据NCBI中绵羊基因组序列，选择绵羊基因组中MSTN基因第二外显子和第三外显子作为靶位点设计sgRNA-1M、sgRNA-2M，ASIP基因的第五外显子作为靶位点设计sgRNA-1A、sgRNA-2A，BCO2基因的第二外显子作为靶位点设计sgRNA-1B、sgRNA-2B其靶位点序列如表1所示，sgRNA序列如表2所示。
表1
表2
2、含有特定特定sgRNA序列的pUC57-T7-gRNA载体的构建：(1)设计并合成识别MSTN基因第二外显子和第三外显子的sgRNA-1M、 sgRNA-2M，识别ASIP基因的第五外显子的sgRNA-1A、sgRNA-2A，识别 BCO2基因的第二外显子的sgRNA-1B、sgRNA-2B；(2)合成后的sgRNA-1M、 sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B成对寡核苷酸分别进行体外退火；(3)将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、 sgRNA-1B、sgRNA-2B通过BsaI位点进行酶切、连接，插入到pUC57-T7-gRNA 中，分别命名为pUC57-T7-sgRNA-1M、pUC57-T7-sgRNA-2M、 pUC57-T7-sgRNA-1A、pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、 pUC57-T7-sgRNA-2B；
针对MSTN、ASIP、BCO2基因的CRISPR/Cas9系统即为：体外转录载体pUC57-T7-sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B和 pST1374-NLS-flag-linker-Cas9。
步骤二：体外转录
利用构建好的载体pUC57-T7-sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B和Cas9mRNA的体外转录载体 pST1374-NLS-flag-linker-Cas9进行以T7启动子介导的体外转录。(1)将 pUC57-T7-sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B分别用 Dra I线性化，pST1374-NLS-flag-linker-Cas9用Age1线性化；(2)用 MEGAshortscript kit(Ambion)试剂盒，按照说明书进行pUC57-T7-sgRNA-1M、 pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、pUC57-T7-sgRNA-2A、 pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B、pST1374-NLS-flag-linker-Cas9 的体外转录；(3)用MEGAClear kit(Ambion)试剂盒，按照说明书进行 pUC57-T7-sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B、Cas9 mRNA的纯化。
步骤三：利用针对MSTN、ASIP、BCO2基因的CRISPR/Cas9系统生产共同敲除MSTN、ASIP、BCO2基因的基因打靶绵羊
1、原核注射及胚胎移植
从经过同期发情后自然交配的供体母绵羊体内通过手术收集处于单细胞阶段的胚胎，利用显微注射仪将预混好的sgRNA-1M、sgRNA-2M、sgRNA-1A、 sgRNA-2A、sgRNA-1B、sgRNA-2B、Cas9mRNA混合物(混合后终浓度为 Cas9mRNA 20ng/μL，sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、 sgRNA-1B、sgRNA-2B各自5ng/μL)注射入绵羊受精卵的细胞质中。注射后的受精卵转移至Quinn’s Advantage Cleavage Medium(Sage Biopharma，NJ， USA)体外37℃培养24h，然后移植至受体绵羊的输卵管壶腹部与峡部连接处，生产共同敲除MSTN、ASIP、BCO2基因的基因打靶绵羊。
2、共同敲除MSTN、ASIP、BCO2基因的基因打靶绵羊的鉴定
受体母羊生产后，待羔羊长至1周龄后采羔羊的血样，提取羔羊血液基因组DNA。以羔羊血液基因组为模版，针对绵羊MSTN第二外显子和第三外显子、ASIP第五外显子、BCO2第2外显子的引物，序列如表3进行扩增，对获得的PCR产物进行琼脂糖凝胶电泳检测并进行产物体系回收，回收后的 PCR产物进行T7EN1酶切，酶切完后进行电泳检测，检测结果显示两条或多条条带的可能为基因打靶成功；将回收后的PCR产物送测序并进行序列分析，结合T7EN1酶切结果和测序结果分析确定阳性个体；对阳性个体的PCR产物克隆至T载体，转化后挑取阳性克隆再次进行测序，根据测序结果更深一步确定基因敲除成功的阳性个体及阳性个体中碱基的变化方式。
表3
以上虽然已经用一般性说明、具体实施方式对本发明做了详尽的描述，但其仅为本发明的优选实施方式。应当指出，对于本技术领域的普通技术人员来说，在不脱离发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (4)

1.一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其特征在于，包括以下步骤：
(1)构建特异性靶向：MSTN第二外显子和第三外显子、ASIP第五外显子、BCO2第二外显子的sgRNA的体外转录载体；通过体外转录得到针对MSTN第二外显子和第三外显子的sgRNA-1M、sgRNA-2M，针对ASIP第五外显子的sgRNA-1A、sgRNA-2A，针对绵羊BCO2第二外显子的sgRNA-1B、sgRNA-2B；
(2)体外转录Cas9蛋白的体外转录载体，得到Cas9 mRNA；
(3)将步骤(1)和步骤(2)的sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B及Cas9 mRNA纯化后测浓度，然后将sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B与Cas9 mRNA混合，注射入绵羊受精卵细胞质中，然后经体外培养后移植入同种雌性绵羊输卵管中，用于生产共同敲除MSTN、ASIP、BCO2基因的转基因绵羊。
2.根据权利要求1所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其特征在于，sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B与Cas9 mRNA混合后，终浓度为Cas9 mRNA 20ng/μL、sgRNA-1M 5ng/μL、sgRNA-2M 5ng/μL、sgRNA-1A 5ng/μL、sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
3.根据权利要求1所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其特征在于，所述sgRNA-1M、sgRNA-2M、sgRNA-1A、sgRNA-2A、sgRNA-1B、sgRNA-2B的表达载体为pUC57-T7-gRNA，Cas9蛋白的体外转录载体为pST1374-NLS-flag-linker-Cas9。
4.根据权利要求1所述的一种利用CRISPR/Cas9系统共同敲除绵羊MSTN、ASIP、BCO2基因的方法，其特征在于，所述特异性靶向为绵羊MSTN第二外显子和第三外显子的sgRNA-1M、sgRNA-2M，针对绵羊ASIP第五外显子的sgRNA-1A、sgRNA-2A，针对绵羊BCO2第2外显子的sgRNA-1B、sgRNA-2B。
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