CRISPR - Cas9系统介导的工业酵母营养缺陷型菌株构建 <sup>*</sup>

doi:10.13523/j.cb.20191008

中国生物工程杂志

2019, Vol. 39

Issue (10): 67-74 DOI: 10.13523/j.cb.20191008

技术与方法

CRISPR - Cas9系统介导的工业酵母营养缺陷型菌株构建 ^*

陆海燕,李佳蔓,孙思凡,章小毛,丁娟娟,邹少兰(

)

天津大学化工学院系统生物工程教育部重点实验室天津 300072

Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae Strain by CRISPR-Cas9 System

LU Hai-yan,LI Jia-man,SUN Si-fan,ZHANG Xiao-mao,DING Juan-juan,ZOU Shao-lan(

)

Key Laboratory of Systems Bioengineering, Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

全文: PDF(712 KB) HTML

摘要：

以组氨酸营养缺陷型菌株构建为例,在前期分离、诱变和筛选得到的安琪酵母工业菌株衍生菌株K-a中试用CRISPR-Cas9系统进行基因修饰。针对菌株K-a为单倍体、ura3和对潮霉素B敏感的特点,构建了以URA3为选择标记的Cas9表达载体YCplac33-Cas9、以hphNT1为选择标记的gRNA表达载体pRS42H-gHIS1,使用PCR方法合成donor DNA片段。使用醋酸锂法制备感受态细胞K-a (YCplac33-Cas9)、将pRS42H-gHIS1和donor DNA共转化,涂布(CMG ^-URA+300μg/ml潮霉素B)平板,经表型筛选和PCR产物测序证明筛选平板生长菌落为目的转化子K-a (his1)的比例为74.4 %,初步建立了适于利用CRISPR/Cas 9系统进行基因修饰的工业菌株宿主平台和相应的简便、快速进行基因修饰的操作技术流程。

关键词： 酿酒酵母; CRISPR-Cas9; his1; 基因敲除

Abstract:

CRISPR-Cas9 system was first attempted to use in Angel industrial yeast - derived strain K-a, and HIS1 gene knockout was carried out. Because strain K-a is haploid, ura3 and sensitive to hygromycin B, the three key factors were designed as followings:Cas9 expressing vector YCplac33-Cas9 with URA3 marker, gRNA expressing vector pRS42H-gHIS1 with hph NT1 marker, and donor DNA fragment by PCR. By using lithium acetate method, pRS42H-gHIS1 and donor DNA was co-transformed into K-a (YCplac33-Cas9) competent cells and (CMG ^-URA+300μg/ml hygromycin B) plate was used for screening. The target transformants were proved to obtain and the ratio was 74.4% by phenotype screening and PCR product sequencing. So the industrial host platform utilizing CRISPR-Cas9 system was constructed and the simple and rapid gene manipulation protocol was preliminarily formed.

Key words: Saccharomyces cerevisiae CRISPR-Cas9 his1 Gene knockout

收稿日期: 2019-03-16 出版日期: 2019-11-12

ZTFLH:

Q812

基金资助: * 天津市科技计划项目(18YFZCNC01240);国家自然科学基金(31470208)

通讯作者: 邹少兰 E-mail: slzhou@tju.edu.cn

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引用本文:

陆海燕,李佳蔓,孙思凡,章小毛,丁娟娟,邹少兰. CRISPR - Cas9系统介导的工业酵母营养缺陷型菌株构建 ^*[J]. 中国生物工程杂志, 2019, 39(10): 67-74.

LU Hai-yan,LI Jia-man,SUN Si-fan,ZHANG Xiao-mao,DING Juan-juan,ZOU Shao-lan. Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae Strain by CRISPR-Cas9 System. China Biotechnology, 2019, 39(10): 67-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191008 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/67

表1 实验所用引物

表2 实验所用质粒

图1 质粒YCplac33-Cas9和pRS42H-gHIS1

图2 质粒YCplac33-Cas 9构建用片段琼脂糖凝胶电泳图

图3 转化子质粒提取物酶切鉴定琼脂糖凝胶电泳图

表3 宿主菌株K-a潮霉素B抗性水平测试

图4 PCR产物琼脂糖凝胶电泳图

图5 PCR产物琼脂糖凝胶电泳图

图6 YPD和CMG培养基中的生长曲线

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