Optimization of CRISPR-Cas9 system in Eustoma grandiflorum
Xueqi Li,
Fanqi Bu,
Lishan Wang,
Cholmin Kim,
Wanjie Xue,
Man Zhang,
Saneyuki Kawabata,
Qingzhu Zhang,
Yuhua Li,
Yang Zhang
Affiliations
Xueqi Li
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China
Fanqi Bu
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China
Lishan Wang
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China
Cholmin Kim
College of Life Science, Northeast Forestry University, Harbin 150040, China; Branch of Biotechnology, State Academy of Sciences, Pyongyang, the Democratic People’s Republic of Korea
Wanjie Xue
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China
Man Zhang
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China
Saneyuki Kawabata
Institute for Sustainable Agroecosystem Services, Graduate School of Agriculture and Life Science, The University of Tokyo, Tokyo, Japan
Qingzhu Zhang
College of Life Science, Northeast Forestry University, Harbin 150040, China
Yuhua Li
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China; Corresponding author
Yang Zhang
Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China; College of Life Science, Northeast Forestry University, Harbin 150040, China; Corresponding author
Summary: The optimization of the CRISPR-Cas9 system for enhancing editing efficiency holds significant value in scientific research. In this study, we optimized single guide RNA and Cas9 promoters of the CRISPR-Cas9 vector and established an efficient protoplast isolation and transient transformation system in Eustoma grandiflorum, and we successfully applied the modified CRISPR-Cas9 system to detect editing efficiency of the EgPDS gene. The activity of the EgU6-2 promoter in E. grandiflorum protoplasts was approximately three times higher than that of the GmU6 promoter. This promoter, along with the EgUBQ10 promoter, was applied in the CRISPR-Cas9 cassette, the modified CRISPR-Cas9 vectors that pEgU6-2::sgRNA-2/pEgUBQ10::Cas9-2 editing efficiency was 37.7%, which was 30.3% higher than that of the control, and the types of mutation are base substitutions, small fragment deletions and insertions. Finally we obtained an efficient gene editing vector for E. grandiflorum. This project provides an important technical platform for the study of gene function in E. grandiflorum.
