生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 252-264.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0618

• 技术与方法 • 上一篇    下一篇

大肠杆菌双质粒CRISPR-Cas9系统的优化及应用

王凯凯(), 王晓璐, 苏小运(), 张杰()   

  1. 中国农业科学院北京畜牧兽医研究所 动物营养学国家重点实验室,北京 100193
  • 收稿日期:2021-05-11 出版日期:2021-12-26 发布日期:2022-01-19
  • 作者简介:王凯凯,男,硕士研究生,研究方向:微生物代谢工程;E-mail: kkw_2012@163.com
  • 基金资助:
    国家重点研发计划政府间国际科技创新合作重点专项(2019YFE0115000);现代农业产业技术体系(CARS-41)

Optimization and Application of Double-plasmid CRISPR-Cas9 System in Escherichia coli

WANG Kai-kai(), WANG Xiao-lu, SU Xiao-yun(), ZHANG Jie()   

  1. State Key Laboratory of Animal Nutrition,Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193
  • Received:2021-05-11 Published:2021-12-26 Online:2022-01-19

摘要:

近年来,CRISPR-Cas9(clustered regularly interspaced short palindromic repeats)基因编辑系统已经成功应用于多种微生物中。由于CRISPR-Cas9系统仅受PAM(protospacer adjacent motif)位点NGG的限制,因此理论上CRISPR-Cas9系统可以编辑基因组中任何含NGG的位点或基因,但研究发现该系统对影响微生物生长及代谢的关键靶基因改造时会出现效率降低,甚至是无法获得突变株的现象。前期已有大量研究报道了降低CRISPR-Cas9系统脱靶效应的策略,但依然未能有效解决编辑效率降低的问题。因此,本研究通过使用不同拷贝数的质粒调节Cas9、gRNA的表达和同源臂浓度使其协同发挥基因编辑功能,建立了更加高效的双质粒CRISPR-Cas9系统。试验结果表明,该系统对大肠杆菌pfkA(6-phosphofructokinase isozyme 1)、pfkB(6-phosphofructokinase isozyme 2)、zwf(glucose-6-phosphate 1-dehydrogenase)单基因敲除最高效率达到100%;在nagABE基因簇位点通过替换引入甘油激酶基因glpK(glycerol kinase)的效率为10%。相比于单质粒CRISPR-Cas9系统,优化的双质粒系统成功进行了pfkB基因的敲除及glpK基因的整合,且将pfkAzwf基因的敲除效率分别提升了31%和63%。突变株与野生株之间碳源代谢的差异进一步表明基因敲除效率与基因特殊活性相关。结果证明,过量的靶基因同源臂和gRNA的过表达可以有效提升CRISPR-Cas9系统在大肠杆菌中的编辑效率。

关键词: CRISPR-Cas9系统, 大肠杆菌, 基因编辑, 双质粒系统

Abstract:

In recent years,the CRISPR-Cas9(clustered regularly interspaced short palindromic repeats)system has been successfully exploited as versatile genome editing tools in various microorganisms. Because the application of CRISPR-Cas9 system is restricted only by the NGG of PAM(protospacer adjacent motif)sequence,therefore CRISPR-Cas9 system can theoretically edit any site or gene on the genome harboring NGG sequence. However,for the genes that have profound effects on cell growth and metabolism,the editing efficiency would be significantly reduced or even the mutants could not be obtained. Plenty of previous reports have provided valuable strategies to reduce the off-target effects of CRISPR-Cas9 system,but the reduction of editing efficiency is far from being solved. In this study,an efficient double- plasmid CRISPR-Cas9 system was established by using plasmids with different copy numbers to regulate the concentration of homology arms and the expression of Cas9 protein and gRNA,which would make them work more collaboratively for the gene editing purpose. The experimental results showed that the pfkA(6-phosphofructokinase isozyme 1)and pfkB(6-phosphofructokinase isozyme 2)genes in glycolysis pathway and the zwf(Glucose-6-phosphate 1-dehydrogenase)gene in pentose phosphate pathway were successfully deleted using the optimized double-plasmid CRISPR-Cas9 system,with the gene deletion efficiency of up to 100%;the nagABE gene cluster was also successfully replaced by glycerol kinase gene glpK,with the gene integration efficiency of 10%. By contrast,when using the single-plasmid CRISPR-Cas9 system,the pfkB gene deletion and glpK gene integration were successful in double-plasmid CRISPR-Cas9 system,and the efficiency of deleting pfkA and zwf increased by 31% and 63% respectively. The differences in carbon source utilization between mutant and wild-type strains further indicated that the gene editing efficiency was associated with special gene activity. Results from this study demonstrated that the sufficient target gene homology arms and the over-expression of gRNA may efficiently enhance the gene editing efficiency of CRISPR-Cas9 system.

Key words: CRISPR-Cas9 system, Escherichia coli, gene editing, double-plasmid system