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- Title
Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.
- Authors
Wolabu, Tezera W.; Park, Jong-Jin; Chen, Miao; Cong, Lili; Ge, Yaxin; Jiang, Qingzhen; Debnath, Smriti; Li, Guangming; Wen, Jiangqi; Wang, Zengyu
- 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. 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.
- Subjects
CRISPRS; GENOME editing; ARABIDOPSIS; MEDICAGO; MEDICAGO truncatula; HOST plants; SEQUENCE analysis
- Publication
Planta: An International Journal of Plant Biology, 2020, Vol 252, Issue 2, p1
- ISSN
0032-0935
- Publication type
Article
- DOI
10.1007/s00425-020-03415-0