Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice

Jinpeng Zou; Xiangbing Meng; Zhengyuan Hong; Yuchun Rao; Kejian Wang; Jiayang Li; Hong Yu; Chun Wang

doi:10.1016/j.tibtech.2024.10.012

Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice

Trends Biotechnol. 2024 Nov 12:S0167-7799(24)00294-4. doi: 10.1016/j.tibtech.2024.10.012. Online ahead of print.

Authors

Jinpeng Zou¹, Xiangbing Meng², Zhengyuan Hong³, Yuchun Rao⁴, Kejian Wang⁵, Jiayang Li⁶, Hong Yu⁷, Chun Wang⁸

Affiliations

¹ State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China.
² Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
³ State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China; College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
⁴ College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
⁵ State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China; Key Laboratory of Gene Editing Technologies (Hainan), Ministry of Agriculture and Rural Affairs, Sanya 572025, China.
⁶ Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; Yazhouwan National Laboratory, Sanya 572024, China.
⁷ Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; Yazhouwan National Laboratory, Sanya 572024, China. Electronic address: [email protected].
⁸ State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China. Electronic address: [email protected].

PMID: 39537536
DOI: 10.1016/j.tibtech.2024.10.012

Abstract

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^S627I 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.

Keywords: multiplex gene editing; precise editing; random mutation; transgene-free.