# Multiplex Gene Editing and Effect Analysis of Yield, Fragrance, and Blast Resistance Genes in Rice

## TL;DR

This study uses CRISPR/Cas9 to edit multiple genes in rice, improving yield, fragrance, and blast resistance in one go.

## Contribution

A novel multiplex CRISPR/Cas9 vector was developed to simultaneously edit five genes in rice for improved traits.

## Key findings

- Simultaneous editing of five genes achieved 9.38% efficiency in T0 plants.
- Edited rice lines showed increased grain number, grain length, and 2-AP fragrance content.
- Rice blast resistance was significantly enhanced in edited lines without affecting other traits.

## Abstract

Background: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement. Methods: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2) and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed using the Agrobacterium-mediated method with the japonica rice variety Xin Dao 53 as the recipient. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, thousand-grain weight, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T3 generations. Results: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. In the T3 generation, compared with the wild-type (WT), the edited homozygous lines showed increased grain number per panicle (14.60–25.61%), increased grain length (7.39–11.16%), increased grain length–width ratio (8.37–13.02%), increased thousand-grain weight (3.79–9.15%), a 42–64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits. Conclusions: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.