CN111733159A - 用于猪MBP基因敲除的sgRNA组合物及用途 - Google Patents
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Abstract
本发明公开了用于猪MBP基因敲除的sgRNA组合物及用途，所述sgRNA组合物包括分别靶向猪MBP基因外显子1和外显子6的sgRNA序列，通过使用能够表达所述sgRNA及Cas9蛋白的载体可直接获得大片段基因敲除细胞系，本方法操作简单，与传统的基因修饰方法时间更短，效率更高。通过此方法获得的猪MBP基因敲除细胞系可直接用于体细胞克隆获得基因修饰动物猪，猪由于在体型及模拟人类神经系统上相较于小鼠更有优势，因此在今后相关疾病的致病机制及疾病治疗方案研究方面发挥更大的作用。
Description
技术领域
本发明属于基因编辑领域，涉及用于猪MBP基因敲除的sgRNA组合物及用途。
背景技术
现在有一种非常常用的髓鞘缺失类疾病模型鼠，叫shiverer mouse，这种模型鼠的髓鞘碱性蛋白基因即MBP(Myelin basic protein)基因大片段缺失，即从内含子1到外显子6的基因缺失，只剩包括外显子1及12.4-14.4kb的内含子序列。由于MBP基因的大片段缺失导致中枢神经系统髓鞘没有髓鞘细胞。这种小鼠，杂合正常，纯合小鼠一般出生12天左右即开始出现震颤，并且随着年龄的增长，症状加剧，一般在50-100天左右死亡。目前被广泛应用神经生物学，髓鞘缺失类相关疾病的研究。
由于小鼠的体型及神经系统与人类相差较大，并不能很好的模拟人类的相关疾病特征。猪由于在体型上与人类更为相似，且有文章表明猪在模拟人类神经系统疾病上相较于小鼠更有优势。因此，我们拟利用高效的CRISPR/Cas9基因编辑技术和体细胞核移植技术制备髓鞘缺失猪模型，然后再以此模型为基础进行细胞治疗，希望能为相关疾病的细胞治疗和基因治疗奠定基础。
发明内容
针对上述问题，本发明的目的是提供一种高效且简单的方法获得MBP基因大片段敲除猪成纤维细胞，同时本发明还提供相应的制备方法和检测手段。
为实现上述目的，本发明采取的技术方案为：用于猪MBP基因敲除的sgRNA组合物，所述组合物包括sgRNA1和sgRNA2；所述sgRNA1和sgRNA2的核苷酸序列从5’到3’方向如SEQID NO.1和SEQ ID NO.2所示。
本发明还要求保护所述的sgRNA组合物在构建敲除猪MBP基因的sgRNA表达载体组合物中的应用。
本发明还要求保护用于敲除猪MBP基因的sgRNA表达载体组合物，所述表达载体包括表达载体1和表达载体2；1、所述表达载体包括表达载体1和表达载体2；所述表达载体1含有如SEQ ID NO.1所示的序列，所述表达载体2含有如SEQ ID NO.2所示的序列。
作为本发明的优选实施方式，所述表达载体组合物的载体骨架为pX459。
本发明还要求保护所述表达载体组合物的构建方法，包括如下步骤：
(1)合成所述sgRNA对应的正义链和反义链；
(2)酶切载体骨架，并回收酶切产物；
(3)分别将步骤(1)所述sgRNA对应的正义链和反义链进行退火得到双链；
(4)将步骤(3)的双链分别连接到步骤(2)的酶切产物上，转化鉴定，即得所述表达载体组合物。
所述sgRNA的正义链和反义链包含对应所述载体酶切位点的序列，与酶切后的载体末端互补，从而连入载体中。
当载体骨架为pX459，骨架通过BbsI进行酶切，酶切产物的大小约为9162bp；sgRNA1对应的正义链、反义链如SEQ ID NO.3和SEQ ID NO.4所示，sgRNA2对应的正义链、反义链如SEQ ID NO.5和SEQ ID NO.6所示(分别含有BbsI酶切序列)，形成与酶切产物连接的具有BbsI粘性末端的双链DNA片段。
若使用其他载体，根据不同载体的图谱确定酶切位点、sgRNA正义链、反义链上用于连接的酶切位点序列，以及回收产物的大小。
本发明还要求保护所述sgRNA组合物、所述表达载体组合物在制备MBP基因敲除的细胞系或动物模型中的用途。
作为本发明的优选实施方式，所述细胞为猪成纤维细胞。
本发明还要求保护制备MBP基因敲除细胞的方法，所述方法包括将所述的表达载体组合物转入细胞中。
若使用的载体上未包含Cas9蛋白，则需要同时进行Cas9蛋白载体的转染，或使用预先构建好的稳定表达Cas9蛋白的细胞。本发明优选的载体pX459载体上含有Cas9的编码序列，转入细胞后可表达Cas9蛋白以及sgRNA，从而达到基因敲除的目的。
作为本发明的优选实施方式，所述将表达载体组合物转入细胞的方法为电转。
作为本发明的优选实施方式，所述方法还包括进行抗性细胞的筛选。
由于载体上含有相应的抗性基因(一定野生型的细胞中不含有)，通过在培养基中加入抗性基因相应的药物，可对含有相关基因(即为成功转入载体)的细胞进行筛选，从而得到更高纯度的目的细胞。优选载体pX459中含有嘌呤霉素抗性标记，可用适当浓度的嘌呤霉素进行细胞筛选。
作为本发明的优选实施方式，所述方法还包括目标细胞的单克隆细胞的培养。
通过调整细胞培养密度，可将得到筛选后的具有单一基因型的单克隆细胞。
进一步地，通过提取单克隆细胞的DNA进行测序，可鉴别各单克隆中MBP基因的敲除情况。
本发明还要求保护所述方法制备的MBP基因敲除细胞。
作为本发明的优选实施方式，所述细胞为猪成纤维细胞。
所述猪成纤维细胞可用于制备转基因克隆猪。
本发明还要求保护一种MBP基因敲除的细胞或动物模型在用于治疗MBP基因缺陷引起的疾病的药物筛选中的用途，所述MBP基因敲除的细胞或动物模型通过所述表达载体组合物制备得到。
更优选地，所述细胞或动物模型为含有MBP基因大片段缺少的纯合子细胞或动物模型。
本发明所述方法采用的是两条分别靶向猪MBP基因外显子1和外显子6的sgRNA序列，通过筛选(更佳为通过单次细胞筛选)即可获得大片段基因敲除细胞系，本方法操作简单，与传统的基因修饰方法如Cre-loxP(时间更短)，显微注射(无嵌合体)更加高效。通过此方法获得的猪MBP基因敲除细胞系可直接用于体细胞克隆获得基因修饰动物猪，猪由于在体型及模拟人类神经系统上相较于小鼠更有优势，因此在今后相关疾病的致病机制及疾病治疗方案研究方面发挥更大的作用。
附图说明
图1为本发明方法构建敲除MBP基因的猪成纤维细胞Sanger测序结果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1用于猪MBP基因敲除的sgRNA组合物及其表达载体
1.Cas9靶位点的选择
Cas9靶位点包含20个碱基，紧邻靶点3’端的3个碱基构成PAM区，PAM区要求序列为NGG(N为任意碱基)。根据该原则设计，针对猪MBP基因第1外显子和第6外显子分别设计一条sgRNA，从5’-3’方向序列如下：
针对第1外显子设计的sgRNA序列：GAGGCACGGATCAAAGTACC(SEQ ID NO.1)；相应的正义链序列和反义链序列为：
1F：CACCGGAGGCACGGATCAAAGTACC(SEQ ID NO.3)，
1R：AAACGGTACTTTGATCCGTGCCTCC(SEQ ID NO.4)；
针对第6外显子设计的sgRNA序列：GAGGACGGGACAGCCGCTCC(SEQ ID NO.2)；相应的正义链序列和反义链序列为：
2F：CACCGGAGGACGGGACAGCCGCTCC(SEQ ID NO.5)，
2R：AAACGGAGCGGCTGTCCCGTCCTCC(SEQ ID NO.6)；
加粗部分包括BbsI酶切位点。
2.CRISPR Cas9质粒构建
(1)酶切骨架质粒：骨架质粒为PX459 V2.0(addgene,62988)，酶切体系如下表1，37℃酶切5h左右，回收之前先取5μL产物跑胶确认酶切完全。
表1酶切体系
| PX459 V2.0 | 3μg |
| BbsI(NEB，R3539S) | 2μL |
| 10×Buffer | 10μL |
| ddH<sub>2</sub>O | 补至100μL |
采用胶回收试剂盒(TIANGEN)回收酶切产物(约9162bp)。
(2)Oligos磷酸化和退火：按下表2分别配制退火体系。
表2退火体系
| Oligo F(100μM) | 1μL |
| Oligo R(100μM) | 1μL |
| 10×T4 Ligation Buffer(NEB) | 1μL |
| T4 PNK(NEB,M0201S) | 0.5μL |
| ddH<sub>2</sub>O | 6.5μL |
退火程序为：37℃30min，95℃5min，接着以5℃/min的速度降温至25℃。
(3)连接：按下表3配制连接体系，16℃连接过夜。
表3连接体系
| 步骤(1)的酶切回收的产物 | 50ng |
| 步骤(2)的oligos(稀释200倍) | 1μL |
| T4 Ligase(NEB) | 0.5μL |
| 10×T4 Ligation Buffer(NEB) | 1μL |
| ddH<sub>2</sub>O | 补至10μL |
(4)转化连接产物，挑取单克隆进行测序(pLKO1.5，测序公司通用引物)，得到连有sgRNA的表达载体组。
实施例2猪MBP基因敲除细胞的筛选
1.嘌呤霉素筛选浓度的确定
猪成纤维细胞生长至80％左右后以1:10传代；24h后，加入含不同浓度嘌呤霉素的新鲜培养基(0-2μg/mL内设置至少5个浓度梯度)。每日显微镜下观察细胞存活比例(嘌呤霉素的最佳作用时间一般在1-4天)，选用加药2-3天杀死所有细胞的最低筛选浓度作为筛选细胞时选用的嘌呤霉素浓度。本实验的嘌呤霉素筛选浓度为800ng/μL。
2.细胞的转染与筛选
(1)在含有10％FBS的DMEM培养基培养猪成纤维细胞，待细胞长至70-90％左右时以1:2比例进行传代，当传代的细胞长至70-90％左右时，可以用于细胞转染；
(2)通过电转转入实施例1的表达载体组；
(3)转染24h后，根据细胞生长情况分板，将细胞消化后以一定比例分至10cm皿培养板上(一般是细胞均匀分至10cm板上后，在显微镜下观察，4×物镜下一个视野看到10-20个细胞左右最佳)；
(4)分板24h后，将培养基换成含0.8μg/mL嘌呤霉素、10％FBS的DMEM培养基(每天观察细胞，嘌呤霉素一般作用两天即可换回原培养基，若发现10cm板上的细胞过多，两天之后可以继续加入含嘌呤霉素的培养基)；
(5)细胞筛选后10～14天左右在显微镜下可看到细胞克隆，通过克隆环(康宁，3166-8)胰酶消化法挑取细胞克隆，将每个细胞克隆挑取至24孔板中，待24孔细胞克隆长至90％左右时，1：2传代，并取出部分细胞进行PCR，测序鉴定。
鉴定所用的上游引物一般设计在sgRNA位点上游100-200bp处，下游引物一般设计在sgRNA位点下游100-200bp处。本次实验的鉴定引物为：
FP1：GGGAGGGAGGACAACACCTTCA，
RP1：GGGATGTCACTGTCTCCGAGGTA。
PCR扩增条件为：
3.结果：
共获得76个细胞克隆，经测序鉴定，其中大片段基因敲除纯合子克隆有3个，效率为3.95％(3/76)，这3个克隆分别为43号、63号及73号，获得3号为野生型，Sanger测序如图1。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案，而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细地说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 五邑大学
<120> 用于猪MBP基因敲除的sgRNA组合物及用途
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Claims (10)
1.用于猪MBP基因敲除的sgRNA组合物，其特征在于，所述组合物包括sgRNA1和sgRNA2；所述sgRNA1和sgRNA2的核苷酸序列从5’到3’方向如SEQ ID NO.1和SEQ ID NO.2所示。
2.如权利要求1所述的sgRNA组合物在构建敲除猪MBP基因的sgRNA表达载体组合物中的应用。
3.用于敲除猪MBP基因的sgRNA表达载体组合物，其特征在于，所述表达载体包括表达载体1和表达载体2；所述表达载体1含有如SEQ ID NO.1所示的序列，所述表达载体2含有如SEQ ID NO.2所示的序列。
4.如权利要求3所述的表达载体组合物，其特征在于，所述表达载体组合物的载体骨架为pX459。
5.如权利要求3或4所述的表达载体组合物的构建方法，其特征在于，包括如下步骤：
(1)合成如权利要求1所述sgRNA对应的正义链和反义链；
(2)酶切载体骨架，并回收酶切产物；
(3)分别将步骤(1)所述sgRNA对应的正义链和反义链进行退火得到双链；
(4)将步骤(3)的双链分别连接到步骤(2)的酶切产物上，转化鉴定，即得所述表达载体组合物。
6.如权利要求1所述的sgRNA组合物、如权利要求3或4所述表达载体组合物在制备MBP基因敲除的细胞系或动物模型中的用途。
7.制备MBP基因敲除细胞的方法，其特征在于，包括将如权利要求3或4所述的表达载体组合物转入细胞中。
8.如权利要求7所述的方法制备的MBP基因敲除细胞。
9.如权利要求8所述的细胞，其特征在于，所述细胞为猪成纤维细胞。
10.一种MBP基因敲除的细胞或动物模型在用于治疗MBP基因缺陷引起的疾病的药物筛选中的用途，其特征在于，所述MBP基因敲除的细胞或动物模型通过如权利要求3或4所述表达载体组合物制备得到。
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