Zou Jinpeng, Meng Xiangbing, Hong Zhengyuan, Rao Yuchun, Wang Kejian, Li Jiayang, Yu Hong, Wang Chun
State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China.
Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Trends Biotechnol. 2025 Feb;43(2):433-446. doi: 10.1016/j.tibtech.2024.10.012. Epub 2024 Nov 12.
In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALS to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.
在分子设计育种中，通过特定的核苷酸修饰同时引入所需的功能基因以及消除调控不良表型性状或农艺成分的基因，需要先进的基因编辑工具。由于编辑效率有限，即使使用高度精确的编辑工具，如碱基编辑（PE），同时编辑多种突变类型也具有挑战性。在此，我们将Cas9切口酶（nCas9）替换为Cas9，在水稻中构建了一种Cas9介导的碱基编辑（Cas9-PE）系统。该系统不仅能够实现精确编辑，还允许进行位点特异性随机突变。此外，利用Cas9-PE的精确性，我们采用共编辑策略建立了一种无转基因的多重基因编辑系统。该策略涉及农杆菌介导的水稻内源乙酰乳酸合成酶基因ALS的精确编辑的瞬时表达，以赋予除草剂双草醚（BS）抗性作为选择标记。本研究为分子设计育种提供了一种通用且高效的多重基因编辑工具。
