The use of gene editing technology to precisely delete genome-specific DNA sequences is very important for plant gene function research and crop breeding. Currently, this technique has only been reported in small quantities in animal or human cells. In plants, although prime editing (PE) can also be achieved in rice protoplasts, the editing efficiency is extremely low and the inheritance is not stable.
近日，北京市农林科学院玉米研究所任雯/杨进孝/赵久然团队在aBIOTECH发表了题为“Efficient and precise genomic deletion in rice using enhanced prime editing”的研究论文，该研究对植物PE工具进行了优化，利用稳定转化系统，在水稻中实现了精准、高效的基因组片段删除。
In order to achieve the precise deletion of the target genome fragments, three technical strategies such as PE3, PDel, and PDel/Syn were tried: first, based on the previous monocotyledon gene editing, the researchers fused reverse transcriptase (M-MLV) to the N-terminus of Cas9 protein and introduced the engineered pegRNA (epegRNA) of the structured RNA motif at the 3' end, and constructed a PE3 system to achieve precise deletion of 25-60 bp at six test sites. On this basis, a PDel strategy was designed, which included pegRNAs of positive and negative DNA strands of target deletion fragments. The PDel strategy significantly increased the average editing efficiency of the above six test loci to 55.8%, while the InDel decreased to 12.2%, and the homozygous editing events also increased.
In order to achieve precise deletion of longer fragments, the PDel strategy was further designed, and the results showed that the PDel strategy could achieve precise deletion of 50-2000 bp length. Although the PDel strategy can achieve efficient and precise deletion editing of rice genomes, some regions cannot be precisely edited due to the limitation of PAM positions in pegRNA pairs. For example, when the RT template contains sequences that are homologous to the region of interest, the efficiency of precise editing of the target fragment is significantly reduced compared to the non-homologous sequences. Therefore, in this study, a PDel/Syn strategy was designed, and multiple synonymous base mutations were introduced into the RT template, which achieved precise deletion events in a wider range and increased the proportion of homozygous mutations.
Fig.1 PE3, PDel, and PDel/Syn strategies to achieve genome-accurate deletion in rice
Overall, this study achieved precise gene fragment deletion in rice through PDel and PDel/Syn strategies, which provided a new tool for functional gene research and gene editing breeding, which was helpful for the fine study of promoter cis elements or protein functional domains, and provided more options for promoting the wide application of gene editing technology in agricultural production and the in-depth study of plant gene function.
Liu Mengyuan, Dr. Zhang Xiang and Xu Wen are the co-first authors of the paper, associate researcher Ren Wen, and researcher Yang Jinxiao and Zhao Jiuran are the corresponding authors. Liu Ya, researcher Liu Xinxiang and Kang Guiting, scientific research assistant of the Institute of Maize, Beijing Academy of Agriculture and Forestry Sciences, have guided or participated in related research work. The research was supported by Beijing Scholars, the Innovation Capacity Building Project of the Beijing Academy of Agriculture and Forestry Sciences, and the Postdoctoral Fund of the Beijing Academy of Agriculture and Forestry Sciences.
引用本文：Liu, M., Zhang, X., Xu, W. et al. Efficient and precise genomic deletion in rice using enhanced prime editing. aBIOTECH (2024). https://doi.org/10.1007/s42994-024-00153-9
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