# Turbo‐charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond

## TL;DR

Multiplex CRISPR editing is transforming crop improvement by enabling precise, simultaneous modification of multiple genes and traits.

## Contribution

The paper reviews recent innovations in multiplex CRISPR editing and their impact on polygenic trait engineering in crops.

## Key findings

- Multiplex editing enables efficient trait stacking and de novo domestication in plants.
- High-throughput sequencing improves detection of complex editing outcomes like structural rearrangements.
- Computational tools and synthetic biology-compatible workflows are advancing scalability and precision.

## Abstract

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene‐free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High‐throughput sequencing technologies, including long‐read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements—often missed by standard genotyping—when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user‐friendly, synthetic biology‐compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue‐specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next‐generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

Multiplex CRISPR editing is revolutionizing plant biotechnology by enabling simultaneous, precise modification of multiple genetic targets. This review highlights recent applications in trait stacking, functional genomics, and de novo domestication. Ongoing advances in CRISPR reagents, vector design, high‐throughput sequencing for mutation mapping, and computational tools are enhancing efficiency and scalability. As these toolkits evolve, multiplex editing is emerging as a foundational platform for next‐generation crop improvement.