Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

Frontiers in Plant Science

卷:11

Maofeng Chai¹  Zeng-Yu Wang¹  Lili Cong¹  Juan Sun¹  Zhipeng Liu²  Qinyan Bao²  Tezera W. Wolabu³  Miao Chen³  Jong-Jin Park³  Yaxin Ge³  Bin Xu³ 

[1] College of Grassland Science, Qingdao Agricultural University, Qingdao, China;

[2] College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China;

[3] Noble Research Institute, Ardmore, OK, United States;

关键词: alfalfa;    genome editing;    CRISPR/Cas9;    multiplex;    mutagenesis;    outcrossing;   

DOI  :  10.3389/fpls.2020.01063

来源: DOAJ

【 摘 要 】

Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

【 授权许可】

Unknown