Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct

PeerJ

article

Sen Zhang¹  Shaoping Wu¹  Chunhua Hu¹  Qiaosong Yang¹  Tao Dong¹  Ou Sheng¹  Guiming Deng¹  Weidi He¹  Tongxin Dou¹  Chunyu Li¹  Chenkang Sun¹  Ganjun Yi¹  Fangcheng Bi¹ 

[1] Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization ,(Ministry of Agriculture and Rural Affairs), Guangdong Key Laboratory of Tropical and Subtropical Fruit Tree Research, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences;College of Horticulture and Forestry Sciences, Huazhong Agricultural University;College of Life Sciences, Zhaoqing University;College of Life Sciences, South China Agricultural University

关键词: CRISPR/Cas9;    Genome editing;    Banana;    Vector optimization;   

DOI  :  10.7717/peerj.12664

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species’ triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

【 授权许可】

CC BY