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

• DOI: 10.1007/s00425-020-03415-0
• Publication Date: 2020-07-08T13:03:43Z
• Authors: Tezera W. Wolabu; Jong-Jin Park; Miao Chen; Lili Cong; Yaxin Ge; Qingzhen Jiang; Smriti Debnath; Guangming Li; Jiangqi Wen; Zengyu Wang
• Keywords: Gene Editing; 2. Zero hunger; 0301 basic medicine; Short Communication; Homozygote; Arabidopsis; Agrobacterium; 03 medical and health sciences; Phenotype; Medicago truncatula; Mutation; CRISPR-Cas Systems; Promoter Regions, Genetic; Alleles

Abstract Main conclusion An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula.

Abstract 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.