Huang Xiaoen, Wang Yuanchun, Wang Nian
Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL, United States.
Front Genome Ed. 2022 Feb 28;4:852867. doi: 10.3389/fgeed.2022.852867. eCollection 2022.
Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene from TATA to CACA in grapefruit () and sweet orange (). TATA-edited plants are resistant to the canker pathogen subsp. (). In addition, CBE was successfully used to edit the () gene in citrus. -edited plants were resistant to the herbicide chlorsulfuron. Two -edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The gene was undetectable in the herbicide-resistant citrus plants. This indicates that the edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.
碱基编辑器，如腺嘌呤碱基编辑器（ABE）和胞嘧啶碱基编辑器（CBE），为精确的基因组编辑提供了替代方法，不会产生双链断裂（DSB），从而避免了由DNA修复引起的基因组不稳定风险和不可预测的结果。碱基编辑器介导的柑橘精确基因编辑尚未见报道。在此，我们已成功应用ABE在葡萄柚（）和甜橙（）中将溃疡病易感基因启动子区域的TATA框从TATA编辑为CACA。经TATA编辑的植物对溃疡病病原菌亚种（）具有抗性。此外，CBE已成功用于编辑柑橘中的（）基因。经编辑的植物对除草剂氯磺隆具有抗性。尽管转化的起始构建体包含GFP表达盒，但两株经编辑的植物未显示绿色荧光。在抗除草剂的柑橘植物中未检测到该基因。这表明经编辑的植物不含转基因，代表了首例使用CRISPR技术的无转基因基因编辑柑橘。总之，我们已成功应用碱基编辑器进行柑橘精确基因编辑。CBE碱基编辑器已通过瞬时表达用于产生无转基因柑橘。
