Loss-function mutants of OsCKX gene family based on CRISPR-Cas systems revealed their diversified roles in rice.

Cytokinin (CTK) is an important plant hormone that promotes cell division, controls cell differentiation, and regulates a variety of plant growth and development processes. Cytokinin oxidase/dehydrogenase (CKX) is an irreversible cytokinin-degrading enzyme that affects plant growth and development by regulating the dynamic balance of CTKs synthesis and degradation. There are presumed 11 members of the CKX gene family in rice (Oryza sativa L.), but limited members have been reported. In this study, based on CRISPR-Cas9 and CRISPR-Cas12a genome-editing technology, we established a complete set of OsCKX1-OsCKX11 single-gene mutants, as well as double-gene and triple-gene mutants of different OsCKXs gene combinations with high similarity. The results revealed that CRISPR-Cas12a outperformed Cas9 to generate biallelic mutations, multi-gene mutants, and more diverse genotypes. And then, we found, except the reported OsCKX2, OsCKX4, OsCKX9 and OsCKX11, OsCKX5, OsCKX6, OsCKX7, and OsCKX8 also had significant effects on agronomic traits such as plant height, panicle size, grain size, and grain number per panicle in rice. In addition, the different loss-of-function of the OsCKX genes also changed the seed appearance quality and starch composition. Interestingly, by comparing different combinations of multi-gene mutants, we found significant functional redundancy among OsCKX gene members in the same phylogenetic clade. These data collectively reveal the diversified regulating capabilities of OsCKX genes in rice, and also provide the valuable reference for further rice molecular breeding.