Generation of early-flowering Chinese cabbage (Brassica rapa spp. pekinensis) through CRISPR/Cas9-mediated genome editing
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The CRISPR system enables us to induce precisely targeted mutations in a plant genome. The widely used CRISPR system is composed of a Cas9 protein derived from Streptococcus pyogenes (SpCas9) and a target site-specific guide RNA. In this study, we successfully generated the early-flowering Chinese cabbage (Brassica rapa spp. pekinensis), which is one of the most important vegetables in the world. To generate early-flowering B. rapa without requiring vernalization, we designed seven guide RNAs which target B. rapa homologous genes to the Arabidopsis thaliana FLOWERING LOCUS C (FLC). We first examined the indel mutation efficacy of the designed guide RNAs in protoplasts isolated from young leaves of Kenshin (an inbred line of B. rapa). After selecting four guide RNAs, genome-edited plants were established by delivering the plant binary vectors harboring SpCas9 along with respective guide RNAs into B. rapa hypocotyl explants. In the T0 generation, we found BraFLC2 and BraFLC3 double knockout lines with the indel efficiency of 97.7% and 100%, respectively. The simultaneous mutations of both BraFLC2 and BraFLC3 were inherited in T1 generations with 100% of indel efficiency. The edited lines obtained showed an early-flowering phenotype that did not depend on vernalization. This study provides a practical gene-editing protocol for Chinese cabbage and verifies the function of its multi-copy BraFLC genes.
KeywordsBraFLC Brassica rapa CRISPR Heritable mutation Protoplast Vernalization
SYJ, HK, YPL, J-SK, and S-GK designed the experiments; SYJ, HA, and YDP performed the plant transformation and tissue culture; SYJ, K-HW, HK, and JR analyzed the indel mutations and maintained transformant lines; SYJ and S-GK designed the guide RNA and made vector constructs; SYJ and S-GK mainly wrote the manuscript; YO, GS, HK, YPL, and S-GK contributed to the revision of the manuscript. This work was supported by Institute for Basic Science (IBS-R021-D1), the Basic Science Research Program of the National Research Foundation of Korea, funded by the Korean government (MSIT) [2017R1C1B5076421/ 2018R1A2B6006233] to H.K, the Next-Generation BioGreen 21 Program (PJ01322603) provided by the Rural Development Administration to YO and S-GK. YPL, SYJ, and GS were supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Golden Seed Project, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (no. 213006-05-2-SB110). GS was partially supported by the Korea Research Fellowship Program (2017H1D3A1A01054325) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, South Korea. The authors declare that they have no competing interests.
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