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Cytokinins play key roles in plant growth and development, and hence their biosynthesis and degradation have been extensively studied. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 CKX genes have been identified to date; however, most of their functions remain unknown. In this study, we comprehensively examined the expression patterns and functions of the CKX s in rice by using CRISPR/Cas9 technology to construct mutants of all 11 genes. The results revealed that the ckx single-mutants and higher-order ckx4 ckx9 mutant lines showed functional overlaps and sub-functionalization. Notably, the ckx1 ckx2 and ckx4 ckx9 double-mutants displayed contrasting phenotypic changes in tiller number and panicle size compared to the wild-type. In addition, we identified several genes with significantly altered expression in both the ckx4 and ckx9 single-mutant and double-mutant plants. Many of the differentially expressed genes were found to be associated with auxin and cytokinin pathways, and cytokinins in the ckx4 ckx9 double-mutant were increased compared to the wild-type. Taken together, our findings provide new insights into the functions of CKX genes in rice growth and may provide the foundations for future studies aimed at improving rice yield. [ABSTRACT FROM AUTHOR]