To create oseif3h mutant, we designed sgRNA using the online tool (http://crispr.hzau.edu.cn/CRISPR/, accessed on 28 April 2021). The sgRNA selection criterion is in close proximity to the transcription start site with high evaluation score. To generate CRISPR/Cas9 binary constructs, we inserted the sgRNA driven by OsU6a pol III promoters into the pCAMBIA1300-based Cas9 vector via a Golden Gate ligation method. The sequencing confirmed plasmids were then transformed into Nipponbare callus to create a stable transgenic line by Agrobacterium-mediated transformation as previously described [36]. Subsequently, the transgenic lines were screened based on the hygromycin resistance and direct sanger-sequencing of the target-containing amplicons. Primers used in this research are listed in Table S1.

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