CN108841845A - 一种带有筛选标记的CRISPR/Cas9载体及其构建方法 - Google Patents
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Abstract
本发明公开了一种带有筛选标记的CRISPR/Cas9载体及其构建方法,其步骤为:以分别带有荧光标记基因Mcherry的与35S启动子的质粒为模板,分别扩增Mcherry与35S片段,再通过融合PCR将35S与Mcherry扩增成一个35S+Mcherry的大片段,经电泳、切胶回收目的条带35S+Mcherry;组装重组质粒PHCE‑35S+Mcherry,转化大肠杆菌感受态细胞后提取重组质粒,经PCR鉴定为阳性重组质粒后送样测序鉴定。本发明载体构建只需通过三步PCR,简单易行,无需对酶切载体与PCR产物纯化回收,组装效率高。
Description
技术领域
本发明属于基因工程技术领域,具体地说,涉及一种带有筛选标记的CRISPR/Cas9载体及其构建方法。
背景技术
CRISPR/Cas9基因组定向编辑技术是近几年发展起来的对基因组进行定向精确修饰的一种技术。通过将外源的DNA导入受体细胞染色体的特定位点上,从而特异地改造基因组,研究基因的功能。该技术可以对基因组中的靶位点进行缺失、敲入、核苷酸修正等操作。2013年,科学家第一次将CRISPR/Cas9应用到人类和小鼠细胞系中对基因进行敲除,随后人们在模式植物和其他农作物中也成功获得了应用,经过改造的CRISPR/Cas9系统也迅速地被应用到拟南芥、烟草、高粱、水稻、小麦、玉米等不同植物基因组的定向编辑研究中,并且获得较高的诱导突变率和可稳定遗传的基因组编辑植株。相对于转基因技术,CRISPR/Cas9系统具有操作简单、快捷、不需要巨大的资金投入、在遗传编辑之后不留下转基因的痕迹,无须引用外源基因,因而生物安全性高,不具有转基因争议。
Mcherry是一种来自于蘑菇珊瑚(mushroom coral)的红色荧光蛋白,常用于标记和示踪某些分子和细胞组分。本研究通过35S启动红色荧光标记基因Mcherry的表达,可实现在植物转化后对阳性转化植物的形态学标记筛选,从而提高基因编辑效率。
发明内容
有鉴于此,本发明提供了一种带有筛选标记的CRISPR/Cas9载体及其构建方法。
为了解决上述技术问题,本发明公开了一种带有筛选标记的CRISPR/Cas9载体的构建方法,包括以下步骤:
(1)设计35S-F、35S-R、Mcherry-F、Mcherry-R和测序引物:
(2)以分别带有荧光标记基因Mcherry的与35S启动子的质粒为模板,对Mcherry与35S片段进行PCR扩增;
(3)PHCE-Cas9质粒线性化;
(4)35S+Mcherry和PHCE-Cas9质粒重组;
(5)重组载体PHCE-35S+Mcherry转化;
(6)PHCE-35S+Mcherry质粒提取并送样测序,制备得到带有筛选标记的CRISPR/Cas9载体。
可选地,所述的35S-F、35S-R、Mcherry-F、Mcherry-R和测序引物的核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.5所示。
可选地,所述的PCR扩增回收35S启动子和Mcherry基因具体为:
(2.1)第一轮PCR扩增和回收:以带有Mcherry质粒为模板,Mcherry-F和Mcherry-R为引物PCR扩增Mcherry基因;以带有35S质粒为模板,35S-F和35S-R为引物PCR扩增35S启动子基因;扩增结束后将PCR产物以120V电压跑电泳,待loading buffer中的指示剂分开距离适当后停止电泳,在紫外凝胶成像系统中观察条带大小并拍照记录,确定是目的条带后,割胶,回收目的基因;分别标记为“35S”和“Mcherry”。
(2.2)第二轮PCR扩增和回收:以第一轮PCR回收的目的基因“35S”和“Mcherry”为模板,35S-F和Mcherry-R为引物进行重叠PCR;PCR完成后配胶跑电泳,在凝胶成像系统中观察条带大小,符合目的片段大小后割胶回收,命名为“35S+Mcherry”。
可选地,所述的第一轮PCR扩增的Mcherry基因的扩增体系为:PHCE-Mcherry质粒1微升,Mcherry-F 1微升,Mcherry-R 1微升,Prime STAR max 25微升,ddH2O 22微升,35S启动子基因的扩增体系为:PHCE-35S质粒1微升,35S-F 1微升,35S-R 1微升,Prime STAR max25微升,ddH2O 22微升,所述的第二轮PCR扩增的扩增体系为:35S 1微升,Mcherry 1微升,35S–F 1微升,Mcherry-R 1微升,Prime STAR max 25微升,ddH2O 22微升,第一轮PCR扩增和第二轮PCR扩增的扩增程序为:94℃4min,40×(94℃30S,55℃30S,72℃45S),72℃2min。
可选地,所述的PHCE-Cas9质粒线性化具体为:
(3.1)使用MfeⅠ酶切PHCE-Cas9质粒,使其线性化;
(3.2)将用MfeⅠ酶切开的PHCE-Cas9质粒同未酶切的PHCE-Cas9的质粒一起跑电泳,对比电泳条带大小,检测酶切是否完全。
可选地,所述的MfeⅠ酶的酶切体系为:PHCE-Cas9质粒3微升,缓冲液2微升,MfeⅠ酶0.4微升,ddH2O 14.6微升。
可选地,所述的35S+Mcherry和PHCE-Cas9质粒重组具体为:将重叠PCR产物割胶回收后的目的片段“35S+Mcherry”与酶切完全的线性化PHCE-Cas9质粒进行重组,构建重组的“PHCE-35S+Mcherry”质粒;重组体系为:酶切后的PHCE-Cas9质粒0.3微升,35S+Mcherry1.2微升,重组酶3微升;重组反应条件为:50℃,60min。
可选地,所述的重组载体PHCE-35S+Mcherry转化具体为:将重组的质粒转化DH5a感受态细胞,然后涂布于有抗性的平板上,待平板上长出菌粒后挑取4个单菌落摇菌,用Taq酶做菌液PCR检测,再挑取PCR检测有条带的菌种扩大培养。
可选地,所述的菌液PCR扩增体系为:模板0.5微升,35S-F 1微升,Mcherry-R 1微升,dNTP 1微升,buffer 2微升,Taq酶0.5微升,ddH2O 14微升。
与现有技术相比,本发明可以获得包括以下技术效果:
1)PHCE-35S+Mcherry重组质粒是在已有的商品化的质粒上进行改进,在载体的特异酶切位点引物筛选标记基因,并在组成型启动子35S的启动下表达,带有重组质粒的阳性转化子植物带有可视化的筛选标记基因Mcherry,在荧光显微镜下产生红色荧光,可进行可视的阳性转化子的筛选。
2)本发明采用同源重组技术,通过PCR扩增目的条带、回收,载体的酶切线性化,组装等过程将外源目标片段与CRISPR载体同源重组,载体构建只需通过三步PCR,简单易行,无需对酶切载体与PCR产物纯化回收,组装效率高。
当然,实施本发明的任一产品并不一定需要同时达到以上所述的所有技术效果。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本发明的一部分,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1是本发明MfeⅠ酶的酶切位点;
图2是本发明带有筛选标记的CRISPR/Cas9载体的构建结构示意图。
具体实施方式
以下将配合实施例来详细说明本发明的实施方式,藉此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
实施例1一种带有筛选标记的CRISPR/Cas9载体,其结构示意图如图2所示,利用PHCE-Cas9为基础载体,在载体的MfeⅠ酶的酶切位点装入35S+Mcherry片段,实现在启动子的调控下表达MCHERRY蛋白作为筛选标记,可实现转化后代的高效可视化筛选。
实施例2一种带有筛选标记的CRISPR/Cas9载体的构建方法,包括如下步骤:
(1)设计引物:
表1本发明所用引物信息
(2)PCR扩增回收35S启动子和Mcherry基因:
第一轮PCR扩增和回收:以带有Mcherry质粒为模板,Mcherry-F和Mcherry-R为引物PCR扩增Mcherry基因,扩增体系(50微升)见表2;以带有35S质粒为模板,35S-F和35S-R为引物PCR扩增35S启动子基因,扩增体系(50微升)如表3。扩增结束后将PCR产物以120V电压跑电泳,待loading buffer中的指示剂分开距离适当后停止电泳,在紫外凝胶成像系统中观察条带大小并拍照记录,35S启动子和Mcherry基因大小分别为452bp与293bp,结合与对照Marker DNA分子大小确定是目的条带后,割胶,回收目的基因。分别标记为“35S”和“Mcherry”。
表2 Mcherry基因PCR扩增体系
表3 35S启动子PCR扩增体系
PCR扩增程序(50微升)如表4。其中变性到退火循环40次。
表4第一轮PCR扩增程序
第二轮PCR扩增和回收:以第一轮PCR回收的目的基因“35S”和“Mcherry”为模板,35S-F和Mcherry-R为引物进行重叠PCR,PCR扩增体系(50微升)如表5。PCR完成后配胶跑电泳,在凝胶成像系统中观察条带大小结合Marker DNA大小判断是否与745bp大小相符,割胶回收,命名为“35S+Mcherry”。
表5“35S+Mcherry”PCR扩增体系
(3)PHCE-Cas9质粒线性化
第一步:使用MfeⅠ酶切PHCE-Cas9质粒,使其线性化。酶切体系(20微升)如表6。注:该酶反应时间过久容易出现星号活性;该酶在CutSmart缓冲液中活性最高能达到100%。
表6 MfeⅠ酶的酶切体系
其中反应条件:37℃,60min。
MfeⅠ酶的酶切位点如图1。
第二歩:将用MfeⅠ酶切开的PHCE-Cas9质粒同未酶切的PHCE-Cas9的质粒一起跑电泳,对比电泳条带大小,酶切完全的线性化条带要比未酶切的条带大,从而检测酶切是否完全。
(4)35S+Mcherry和PHCE-Cas9质粒重组
将重叠PCR产物割胶回收后的目的片段“35S+Mcherry”与酶切完全的线性化PHCE-Cas9质粒进行重组,构建重组的“PHCE-35S+Mcherry”质粒。重组体系如表7。
表7 35S+Mcherry与PHCE-Cas9质粒重组体系
其中反应条件:50℃,60min。
(5)重组载体PHCE-35S+Mcherry转化
将重组的质粒转化DH5a感受态细胞,然后涂布于有抗性的平板上,待平板上长出菌粒后挑取4个单菌落摇菌,用Taq酶做菌液PCR检测,相应体系如表8。再挑取PCR检测有条带的菌种扩大培养。
表8菌液PCR扩增体系
扩增程序(20微升)如表9。其中,变性到延伸循环40次。
表9菌液PCR扩增程序
(6)PHCE-35S+Mcherry质粒提取并送样测序
选取上一步菌液PCR中条带较亮的菌种扩大培养,提取质粒后跑质粒检测提取质粒的浓度并进行PCR验证,PCR扩增体系与条件与菌液PCR相同,经与阳性对照相比,能扩增出目的条带的结果确认无误后送样测序。
上述说明示出并描述了发明的若干优选实施例,但如前所述,应当理解发明并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述发明构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离发明的精神和范围,则都应在发明所附权利要求的保护范围内。
序列表
<110> 广东石油化工学院
<120> 一种带有筛选标记的CRISPR/Cas9载体及其构建方法
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Claims (9)
1.一种带有筛选标记的CRISPR/Cas9载体的构建方法,其特征在于,包括以下步骤:
(1)设计35S-F、35S-R、Mcherry-F、Mcherry-R和测序引物:
(2)以分别带有荧光标记基因Mcherry的与35S启动子的质粒为模板,对Mcherry与35S片段进行PCR扩增;
(3)PHCE-Cas9质粒线性化;
(4)35S+Mcherry和PHCE-Cas9质粒重组;
(5)重组载体PHCE-35S+Mcherry转化;
(6)PHCE-35S+Mcherry质粒提取并送样测序,制备得到带有筛选标记的CRISPR/Cas9载体。
2.根据权利要求1所述的构建方法,其特征在于,所述的35S-F、35S-R、Mcherry-F、Mcherry-R和测序引物的核苷酸序列分别如SEQ ID NO.1- SEQ ID NO.5所示。
3.根据权利要求1所述的构建方法,其特征在于,所述的PCR扩增回收35S启动子和Mcherry基因具体为:
(2.1)第一轮PCR扩增和回收:以带有Mcherry质粒为模板,Mcherry-F和Mcherry-R为引物PCR扩增Mcherry基因;以带有35S质粒为模板,35S-F和35S-R为引物PCR扩增35S启动子基因;扩增结束后将PCR产物以120V电压跑电泳,待loading buffer中的指示剂分开距离适当后停止电泳,在紫外凝胶成像系统中观察条带大小并拍照记录,确定是目的条带后,割胶,回收目的基因;分别标记为“35S”和“Mcherry”;
(2.2)第二轮PCR扩增和回收:以第一轮PCR回收的目的基因“35S”和“Mcherry”为模板,35S-F和Mcherry-R为引物进行重叠PCR;PCR完成后配胶跑电泳,在凝胶成像系统中观察条带大小,符合目的片段大小后割胶回收,命名为“35S+Mcherry”。
4.根据权利要求3所述的构建方法,其特征在于,所述的第一轮PCR扩增的Mcherry基因的扩增体系为:PHCE-Mcherry质粒 1微升,Mcherry-F 1微升,Mcherry-R 1微升,PrimeSTAR max 25微升,ddH2O 22微升,35S启动子基因的扩增体系为:PHCE-35S质粒 1微升,35S-F 1微升,35S-R 1微升,Prime STAR max 25微升,ddH2O 22微升,所述的第二轮PCR扩增的扩增体系为:35S 1微升,Mcherry 1微升,35S–F 1微升,Mcherry-R 1微升,Prime STARmax 25微升,ddH2O 22微升,第一轮PCR扩增和第二轮PCR扩增的扩增程序为:94℃ 4min,40×(94℃ 30S,55℃ 30S, 72℃ 45S),72℃ 2min。
5.根据权利要求1所述的构建方法,其特征在于,所述的PHCE-Cas9质粒线性化具体为:
(3.1)使用MfeⅠ酶切PHCE-Cas9质粒,使其线性化;
(3.2)将用MfeⅠ酶切开的PHCE-Cas9质粒同未酶切的PHCE-Cas9的质粒一起跑电泳,对比电泳条带大小,检测酶切是否完全。
6.根据权利要求5所述的构建方法,其特征在于,所述的MfeⅠ酶的酶切体系为:PHCE-Cas9质粒 3微升,缓冲液 2微升,MfeⅠ酶 0.4微升,ddH2O 14.6微升。
7.根据权利要求1所述的构建方法,其特征在于,所述的35S+Mcherry和PHCE-Cas9质粒重组具体为:将重叠PCR产物割胶回收后的目的片段“35S+Mcherry”与酶切完全的线性化PHCE-Cas9质粒进行重组,构建重组的“PHCE-35S+Mcherry”质粒;重组体系为:酶切后的PHCE- Cas9质粒0.3微升,35S+Mcherry 1.2微升,重组酶 3微升;重组反应条件为:50℃,60min。
8.根据权利要求1所述的构建方法,其特征在于,所述的重组载体PHCE-35S+Mcherry转化具体为:将重组的质粒转化DH5a感受态细胞,然后涂布于有抗性的平板上,待平板上长出菌粒后挑取4个单菌落摇菌,用Taq酶做菌液PCR检测,再挑取PCR检测有条带的菌种扩大培养。
9.根据权利要求8所述的构建方法,其特征在于,所述的菌液PCR扩增体系为:模板 0.5微升,35S-F 1微升,Mcherry-R 1微升,dNTP 1微升,buffer 2微升,Taq酶 0.5微升,ddH2O14微升。
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