CN106282241A - 通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法 - Google Patents
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Abstract
本发明公开了通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法，设计了新的gRNA序列，设计在BMP2a第一个外显子和内含子之间，gRNA序列为GGAGCCCATCACTAGACTCTTGG，酶切为HinfI。与传统敲除基因的技术相比，CRISPR/Cas9技术具有毒性小，准确性高，效率高，成功周期短等特点；所以理论上，使得BMP2a基因更快得被敲除。
Description
技术领域
本发明涉及通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法。
背景技术
CRISPR/Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9***通过将入侵噬菌体和质粒 DNA 的片段整合到CRISPR中，并利用相应CRISPR RNAs（crRNAs）来指导同源序列的降解，从而提供免疫性。此***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（short guide 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（unifying factor），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RNA可连接到sgRNA 的末端，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。相比于传统的Talens等敲基因技术，CRISPR/Cas9具有更高效率，更方便操作，优点如下：
1，只需要合成一个sgRNA就能实现对基因的特异性修饰，Cas蛋白不具有特异性。
2，编码sgRNA的序列不超过100bp,因此比构建TALENs和ZFNs更简单方便。
3，较短的sgRNA序列也避免了超长、高度重复的TALENs编码载体带来的并发症。
中国专利201510582860.5公开了一种通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法。那么能否利用CRISPR/Cas9技术，设计独特的一段PAM区，使得斑马鱼中的BMP2a基因被完美敲除，又不“误伤”其他基因，形成世界上首例BMP2a敲除的斑马鱼？
作为首例BMP2a敲除转基因的模式动物斑马鱼的意义重大，BMP2a是调控铁的主要因素，一旦被敲除，即成功动物模成铁过载的模式动物，可以排除人为因素干预，对于研究铁的表达研究意义重大。
发明内容
本发明要解决的技术问题是提供通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼。
为了解决上述的技术问题，本发明提供如下技术方案。
通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法，包括如下步骤：
（1）设计了新的gRNA序列，设计在BMP2a第一个外显子和内含子之间，gRNA序列为GGAGCCCATCACTAGACTCTTGG，酶切为HinfI；
（2）设计并合成gRNA引物，引物见表1：
表1——CAS9BMP2a的引物序列为设计的P3:
Forward sequence (5’to3’):CCTCTTCAACCTGACCTCCA；
Reverse sequence (5’to 3’):GTCCGTCTGTGGTCCACTTT；
P3：GATCACTAATACGACTCACTATAGGAGCCCATCACTAGACTCTGTTTTAGAGCTAGAAAT；
p4：AAAAGCACCGACTCGGTGCC；
gRNA序列GGAGCCCATCACTAGACTCTTGG；
（3）将设计的引物进行PCR，PCR体系如下：
gRNA- plasmid 10 ng；
P3 1 ul(10 pmol)；
P4 1 ul(10 pmol)；
Buffer 10；
dNTP 8；
KOD 0.5；
ddH2O Up to 100 ul；
PCR反应条件为：95℃预变性3min，进入三步循环（95℃-20s、58℃-20s、72℃-20s共30个循环），然后72℃-10min，最后保温在16℃；电泳检测PCR产物后，进行纯化；
(4) RNA-Free条件下，将gRNA进行体外转录，体系为：
2.5mmol/L NTP 4ul；
10× Reaction Buffer 2ul；
Template DNA 1 1ug(<6ul)；
T7 Enzyme Mix 2ul；
DEPC Water up to 20ul ；
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul ；
以上体系37°C ，1 hour反应完毕，然后进行纯化；
(5) 将前述纯化的mRNA 注射到单细胞的斑马鱼胚胎中，4天后提取RNA,转录DNA进行测序检测；注射体系如下：
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
(6)三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到一定概率的杂合子，将胚胎后进行ＲＮＡ提取并转录成ＤＮＡ送测序查看是否有突变，这时候的ＤＮＡ还是双链的；然后将测序后发现突变的斑马鱼的ｃＤＮＡ和１９Ｔ载体相连接后，将其在培养基中滚珠图板，经过１２－１４小时后，已经连接的质粒会以斑点形式成长，将其挑斑后，得到的是单链的ＤＮＡ，再次送测序，最终得到单链的突变，然后将其培养长大三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到胚胎后查看是否有突变，将突变的斑马鱼挑单克隆测序后养起来；
(7) 经过三个月后性成熟后，第二代的突变成年的雄鱼与雌鱼再次切除尾巴，进行鉴定，详见步骤（6），得到相同突变（缺失几个碱基）的斑马鱼再次交配，从而得到纯合BMP2a敲除的斑马鱼。
作为本发明一优选技术方案，步骤（3）和步骤（4）中的纯化方法包括如下步骤：
(A) 加入体积为1：2-3的水和苯酚/氯仿/异丙醇, 混匀后10,000-15,000 rpm离心5min. 上层移入新的管子，重复该步骤一次；
(B)上层移入新的管子，加入150ul氯仿,离心5 min；
(C)加入1/10体积2.5 M醋酸钠以及2.5体积乙醇,-70C冷冻30min；
(D)离心10,000-15,000rpm在4C 15min；
(E)弃掉溶液留沉淀，加入 200ul 80%乙醇,离心5 min，弃掉溶液，干燥后用10-20µlDEPC H2O溶解。
与传统敲除基因的技术相比，CRISPR/Cas9技术具有毒性小，准确性高，效率高，成功周期短等特点；所以理论上，使得BMP2a基因更快得被敲除。
附图说明
图1为本发明方法的培育杂交图系。
具体实施方式
具体实施方式
此次实施例中所用的引物均为苏州金唯智公司合成。野生型斑马鱼AB品系，苏州大学生物钟研究中心。
具体实验过程：
设计gRNA位点-PCR-纯化-体外转录-纯化-显微注射-鉴定活性-饲养至成年-与野生型交配-检测下一代胚胎是否携带突变位点-饲养-成年后剪尾鉴定出杂合突变体-两杂合体交配得到纯合突变体。
通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法，包括如下步骤：
（1）设计了新的gRNA序列，设计在BMP2a第一个外显子和内含子之间，gRNA序列为GGAGCCCATCACTAGACTCTTGG，酶切为HinfI；
（2）设计并合成gRNA引物，引物见表1：
表1——CAS9BMP2a的引物序列为设计的P3:
Forward sequence (5’to3’):CCTCTTCAACCTGACCTCCA；
Reverse sequence (5’to 3’):GTCCGTCTGTGGTCCACTTT；
P3：GATCACTAATACGACTCACTATAGGAGCCCATCACTAGACTCTGTTTTAGAGCTAGAAAT；
p4：AAAAGCACCGACTCGGTGCC；
gRNA序列GGAGCCCATCACTAGACTCTTGG；
（3）将设计的引物进行PCR，PCR体系如下：
gRNA- plasmid 10 ng；
P3 1 ul(10 pmol)；
P4 1 ul(10 pmol)；
Buffer 10；
dNTP 8；
KOD 0.5；
ddH2O Up to 100 ul；
PCR反应条件为：95℃预变性3min，进入三步循环（95℃-20s、58℃-20s、72℃-20s共30个循环），然后72℃-10min，最后保温在16℃；电泳检测PCR产物后，进行纯化；
(4) RNA-Free条件下，将gRNA进行体外转录，体系为：
2.5mmol/L NTP 4ul；
10× Reaction Buffer 2ul；
Template DNA 1 1ug(<6ul)；
T7 Enzyme Mix 2ul；
DEPC Water up to 20ul ；
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul ；
以上体系37°C ，1 hour反应完毕，然后进行纯化；
(5) 将前述纯化的mRNA 注射到单细胞的斑马鱼胚胎中，4天后提取RNA,转录DNA进行测序检测；注射体系如下：
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
(6)三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到一定概率的杂合子，将胚胎后进行ＲＮＡ提取并转录成ＤＮＡ送测序查看是否有突变，这时候的ＤＮＡ还是双链的；然后将测序后发现突变的斑马鱼的ｃＤＮＡ和１９Ｔ载体相连接后，将其在培养基中滚珠图板，经过１２－１４小时后，已经连接的质粒会以斑点形式成长，将其挑斑后，得到的是单链的ＤＮＡ，再次送测序，最终得到单链的突变，然后将其培养长大三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到胚胎后查看是否有突变，将突变的斑马鱼挑单克隆测序后养起来；
(7) 经过三个月后性成熟后，第二代的突变成年的雄鱼与雌鱼再次切除尾巴，进行鉴定，详见步骤（6），得到相同突变（缺失几个碱基）的斑马鱼再次交配，从而得到纯合BMP2a敲除的斑马鱼。
作为本发明一优选技术方案，步骤（3）和步骤（4）中的纯化方法包括如下步骤：
(A) 加入体积为1：2-3的水和苯酚/氯仿/异丙醇, 混匀后10,000-15,000 rpm离心5min. 上层移入新的管子，重复该步骤一次；
(B)上层移入新的管子，加入150ul氯仿,离心5 min；
(C)加入1/10体积2.5 M醋酸钠以及2.5体积乙醇,-70C冷冻30min；
(D)离心10,000-15,000rpm在4C 15min；
(E)弃掉溶液留沉淀，加入 200ul 80%乙醇,离心5 min，弃掉溶液，干燥后用10-20µlDEPC H2O溶解。
Claims (2)
1.通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法，包括如下步骤：
（1）设计了新的gRNA序列，设计在BMP2a第一个外显子和内含子之间，gRNA序列为GGAGCCCATCACTAGACTCTTGG，酶切为HinfI；
（2）设计并合成gRNA引物，引物见表1：
表1——CAS9BMP2a的引物序列为设计的P3:
Forward sequence (5’to3’):CCTCTTCAACCTGACCTCCA；
Reverse sequence (5’to 3’):GTCCGTCTGTGGTCCACTTT；
P3：GATCACTAATACGACTCACTATAGGAGCCCATCACTAGACTCTGTTTTAGAGCTAGAAAT；
p4：AAAAGCACCGACTCGGTGCC；
gRNA序列GGAGCCCATCACTAGACTCTTGG；
（3）将设计的引物进行PCR，PCR体系如下：
gRNA- plasmid 10 ng；
P3 1 ul(10 pmol)；
P4 1 ul(10 pmol)；
Buffer 10；
dNTP 8；
KOD 0.5；
ddH2O Up to 100 ul；
PCR反应条件为：95℃预变性3min，进入三步循环（95℃-20s、58℃-20s、72℃-20s共30个循环），然后72℃-10min，最后保温在16℃；电泳检测PCR产物后，进行纯化；
(4) RNA-Free条件下，将gRNA进行体外转录，体系为：
2.5mmol/L NTP 4ul；
10× Reaction Buffer 2ul；
Template DNA 1 1ug(<6ul)；
T7 Enzyme Mix 2ul；
DEPC Water up to 20ul ；
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul ；
以上体系37°C ，1 hour反应完毕，然后进行纯化；
(5) 将前述纯化的mRNA 注射到单细胞的斑马鱼胚胎中，4天后提取RNA,转录DNA进行测序检测；注射体系如下：
gRNA 12.5(ng/ul)；
Cas9 300(ng/ul)；
Tris-Hcl 0.2ul；
Phenol-red 0.2ul；
DEPC Water up to 2ul；
(6)三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到一定概率的杂合子，将胚胎后进行ＲＮＡ提取并转录成ＤＮＡ送测序查看是否有突变，这时候的ＤＮＡ还是双链的；然后将测序后发现突变的斑马鱼的ｃＤＮＡ和１９Ｔ载体相连接后，将其在培养基中滚珠图板，经过１２－１４小时后，已经连接的质粒会以斑点形式成长，将其挑斑后，得到的是单链的ＤＮＡ，再次送测序，最终得到单链的突变，然后将其培养长大三个月后性成熟后，将突变的斑马鱼与野生型的斑马鱼杂交，得到胚胎后查看是否有突变，将突变的斑马鱼挑单克隆测序后养起来；
(7) 经过三个月后性成熟后，第二代的突变成年的雄鱼与雌鱼再次切除尾巴，进行鉴定，详见步骤（6），得到相同突变（缺失几个碱基）的斑马鱼再次交配，从而得到纯合BMP2a敲除的斑马鱼。
2.如权利要求1所述的通过CRISPR/Cas9得到敲除bmp2a基因的斑马鱼的方法，其特征为；步骤（3）和步骤（4）中的纯化方法包括如下步骤：
(A) 加入体积为1：2-3的水和苯酚/氯仿/异丙醇, 混匀后10,000-15,000 rpm离心5min. 上层移入新的管子，重复该步骤一次；
(B)上层移入新的管子，加入150ul氯仿,离心5 min；
(C)加入1/10体积2.5 M醋酸钠以及2.5体积乙醇,-70C冷冻30min；
(D)离心10,000-15,000rpm在4C 15min；
(E)弃掉溶液留沉淀，加入 200ul 80%乙醇,离心5 min，弃掉溶液，干燥后用10-20µlDEPC H2O溶解。
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