Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations
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Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations Xiaoen Huang1 · Yuanchun Wang1 · Jin Xu1 · Nian Wang1 Received: 7 June 2020 / Accepted: 28 July 2020 © Springer Nature B.V. 2020
Abstract Key message We have developed multiplex genome editing toolkits for citrus that significantly improve citrusgenome editing efficacy. Abstract CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus. Keywords Citrus · Genome editing · CRISPR/Cas9 · Multiplex · tRNA · Csy4 Citrus is one of the top three fruit crops worldwide and is an important source of many nutrients, such as vitamin C. Genetic improvement of citrus is required to overcome many challenges encountered in citrus cultivation, such as Huanglongbing caused by Candidatus Liberibacter (Bove 2006; Wang et al. 2017). Conventional citrus breeding is a lengthy, laborious and challenging process due to the complex reproductive biology of citrus, including sexual incompatibility, a highly heterozygous nature, nucellar seedlings, male or female sterility and a long juvenile phase (Omura and Shimada 2016). CRISPR-mediated genome editing (Jinek et al. 2012), which has been successfully used to edit the genomes of many plant species(Chen et al. 2019; Li et al. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11103-020-01043-6) contains supplementary material, which is available to authorized users. * Nian Wang [email protected] 1
Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA
2013; Nekrasov et al. 2013; Puchta 2017; Schindele et al. 2018; Shan et al. 2013; Zhang et al. 2019b), has the potential to accelerate the citrus improvement process (Jia and Wang 2014; Weeks 2017; Zhang et al. 2017). For example, Cas9/ sgRNA has been used to generate disease-resistant citrus varieties against bacterial canker disease caused by Xanthomonas citri subsp. citri (Xcc) by modifying either the promoter region or the c
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