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CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot ‘Kurodagosun’ resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot ‘Deep purple’, resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.

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Acknowledgements

The authors thank Prof. Yao-Guang Liu (South China Agriculture University, Guangzhou, China) for providing the plant binary vector pYLCRISPR/Cas9 and the sgRNA plasmids. The research was supported by the National Natural Science Foundation of China (Grant Nos. 31501775; 31872098), Open Project of State Key Laboratory of Crop Genetics and Germplasm Enhancement (Grant No. ZW201710) and Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant No. PAPD).

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  1. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

    Zhi-Sheng Xu, Kai Feng & Ai-Sheng Xiong

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Xu, ZS., Feng, K. & Xiong, AS. CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants. Mol Biotechnol 61, 191–199 (2019). https://doi.org/10.1007/s12033-018-00150-6

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