Plant miRNAs are a class of noncoding RNA with a length of 21–24 nt that play an important role in plant responses to biotic and abiotic stresses. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases in rice. Our previous work showed that osa-miR2118b/n was induced by Xoo infection. However, the biological function of miR2118 has not yet been characterized in experiments. Herein, we constructed MIR2118b OE, as well as single and double mutants of MIR2118b/n using CRISPR/Cas9. Further results showed that osa-MIR2118b OE plants exhibited longer lesion lengths than the wild type after Xoo inoculation, while MIR2118 CRISPR plants exhibited shorter lesion lengths than the wild type after Xoo inoculation. Co-transformation experiments in rice protoplasts indicated that osa-miR2118 negatively regulated the transcripts of three nucleotide-binding sites and leucine-rich repeat (NLR) genes (LOC_Os08g42700.1, LOC_Os01g05600.1, and LOC_Os12g37290.1) which are predicted target genes of miR2118, but not the mutated NLR genes with a 3 bp insertion at the center of the binding sites. The transcriptional level of the three NLR genes was reversed relative to osa-miR2118 in the MIR2118b OE and MIR2118b CRISPR plants. The above results demonstrate that osa-miR2118b/n negatively regulates the resistance to bacterial blight through negatively regulating several NLR genes.

Streptococcus canis Cas9 (ScCas9) is an RNA‐guided endonuclease with NNG protospacer adjacent motif (PAM) specificity whose genome‐editing activity in rice is locus‐dependent. Here we investigated the performance of a ScCas9 variant named Sc⁺⁺ at different NNG PAM sites in the rice genome; Sc⁺⁺ harbors a T1227K mutation and the substitution of a positively charged loop (residues 367–376). Sc⁺⁺ nuclease achieved broader genome editing compared to the original ScCas9, and its nickase improved targeted base editing in transgenic rice plants. Using the high‐efficiency adenine base editor rBE73b, we generated many new OsGS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future. The CRISPR/Sc⁺⁺ system expands the genome‐editing toolkit for rice.