Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula

• Published in: Planta Vol. 252; no. 2; p. 15
• Main Authors: Wolabu, Tezera W., Park, Jong-Jin, Chen, Miao, Cong, Lili, Ge, Yaxin, Jiang, Qingzhen, Debnath, Smriti, Li, Guangming, Wen, Jiangqi, Wang, Zengyu
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.08.2020; Springer Nature B.V
• Subjects: Agriculture; Agrobacterium - genetics; Alfalfa; Alleles; Arabidopsis; Arabidopsis - genetics; Biomedical and Life Sciences; CRISPR; CRISPR-Cas Systems - genetics; Desaturase; Ecology; Efficiency; Forestry; Gene Editing; Genetic modification; Genetic transformation; Genome editing; Genomes; Homozygote; Host plants; Life Sciences; Medicago truncatula - genetics; Mutants; Mutation; Nucleotides; Pds gene; Phenotype; Plant Sciences; Promoter Regions, Genetic - genetics; Sequences; Short Communication; Species; Vectors; Genome editing; Mutation efficiency; CRISPR/Cas9; Promoters; Dicots
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-020-03415-0

Main conclusion An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase ( PDS ) genes were designed. Agrobacterium -mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.