Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula
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SHORT COMMUNICATION
Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula Tezera W. Wolabu1 · Jong‑Jin Park1,2 · Miao Chen1,3 · Lili Cong1,4 · Yaxin Ge1 · Qingzhen Jiang1 · Smriti Debnath1 · Guangming Li1 · Jiangqi Wen1 · Zengyu Wang1,5 Received: 9 April 2020 / Accepted: 23 June 2020 © The Author(s) 2020
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 monoallelic 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. Keywords CRISPR/Cas9 · Dicots · Genome editing · Mutation efficiency · Promoters
Communicated by Dorothea Bartels. Tezera W. Wolabu and Jong-Jin Park authors of equal contribution.
Abbreviations AMGE3 Meiosis-specific promoter AtPDS3 Arabidopsis thaliana PDS EC1.2 Egg cell-specific PAM Protospacer adjacent motif PDS Phytoene desaturase UBQ10 Ubiquitin10
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00425-020-03415-0) contains supplementary material, which is available to authorized users. * Jiangqi Wen [email protected] * Zengyu Wang [email protected] 1
Noble Research Institute, LLC, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA
Present Address: Genome Editing Naturegenic Inc, 1281 Win Hentschel Boulevard, Kurz Purdue Technology Center Suite E‑1251, West Lafayette, IN 47906, USA
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Present Address: Guang Dong Ocean University, Faculty of Agricultural Science, #1 Haida Road, Mazhang, Zhanjiang 524088, Guangdong, China
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Present Address: College of Grassland Science, Qingdao Agricultural University, Changch
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