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

A Cas9-mediated prime editing (Cas9-PE) system was developed with the ability to induce random mutations in rice.A multiplex gene editor for simultaneous precise editing and random mutations was developed in rice.Transgene-free gene editing technology was upgraded via co-editing with the herbicide resistance gene ALSS627I. 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 ALSS627I 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. [Display omitted] This study developed a powerful multiplex gene editor, known as the Cas9-mediated prime editing (Cas9-PE) system, for simultaneous precise editing and site-specific random mutation in rice. Significantly, the herbicide resistance mutation ALSS627I was implemented for co-editing to generate transgene-free gene-edited plants in the T0 generation. The Cas9-mediated prime editing (Cas9-PE) system introduced in this study is currently at a Technology Readiness Level (TRL) of 4, indicating its validation in laboratory settings using stably transformed T0 plants. Although the Cas9-PE system has been demonstrated as a powerful multiplex gene editor achieving simultaneous precise editing and site-specific random mutation in a transgene-free manner in T0 plants, several challenges must be addressed before it can be fully implemented. Importantly, different strategies need to be adopted to improve the efficiency of the Cas9-PE system for transgene-free editing. Moreover, implementing the Cas9-PE system in crop breeding will depend on obtainin