Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.

Multiplex genome editing is a powerful approach for dissecting gene networks and engineering complex traits in crops because it enables the simultaneous modification of multiple genomic loci. However, achieving high editing efficiency across multiple targets remains a significant challenge. To address this, we developed an optimized CRISPR system for rice that combines a monomeric TREX2-SpCas9 fusion with a novel array of tRNA-based gRNA processing elements. The TREX2-SpCas9 fusion significantly enhanced editing performance, resulting in higher editing efficiency, larger deletions, and increased mutation frequencies compared with wild-type SpCas9 and other exonuclease fusions. By systematically evaluating 38 endogenous rice tRNA genes, we identified 13 high-performing candidates, including tRNALeu-1 and tRNAPro-1, that outperformed the widely used tRNAGly and tRNAMet elements, enabling highly efficient processing of multiplexed gRNA arrays. Incorporating these top-performing tRNAs into our system enabled simultaneous editing of up to 29 OsCPK genes in a single rice plant. Furthermore, we demonstrated the cross-species applicability of this platform in the dicot Nicotiana benthamiana using transient expression, where rice-derived tRNA elements facilitated high-efficiency editing. This optimized multiplex gene-editing system provides a robust, scalable platform for accelerating plant functional genomics and engineering complex agronomic traits.