To create oseif3h mutant, we designed sgRNA using the online tool (, 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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