Journal
PLANT MOLECULAR BIOLOGY
Volume 88, Issue 6, Pages 561-572Publisher
SPRINGER
DOI: 10.1007/s11103-015-0342-x
Keywords
CRISPR/Cas9; Genome editing; Targeted mutagenesis; Rice
Categories
Funding
- Ministry of Agriculture, Forestry and Fisheries of Japan [PGE1001]
- CSTI, Cross-ministerial SIP, Technologies for creating next-generation agriculture, forestry and fisheries (funding agency: Bio-oriented Technology Research Advancement Institution, NARO)
- NIAS Strategic Research Fund
Ask authors/readers for more resources
The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T-0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available