Step-by-Step Cloning of gRNA-Targeted OsCYP71A1 for Gene Editing Study

Serotonin, a neurotransmitter in mammals, is also synthesized in rice in response to insect infestation. However, this serotonin production enhances susceptibility to two of the most damaging rice pests-planthoppers and stem borers. The cytochrome P450 gene CYP71A1 in rice encodes tryptamine 5-hydroxylase, the enzyme that converts tryptamine into 5-hydroxytryptamine (serotonin). In wild-type rice, herbivory by planthoppers triggers the production of serotonin and salicylic acid. In contrast, CYP71A1 mutants lacking serotonin biosynthesis exhibit elevated levels of salicylic acid and display enhanced resistance to insect attack. To investigate the role of CYP71A1 in pest resistance, we aimed to disrupt this gene using the CRISPR-Cas9 gene editing system. To achieve targeted modification of the OsCYP71A1 gene, we utilized the CRISPR/Cas9 system, specifically designing a 20-bp gRNA to maximize on-target precision while mitigating potential off-target effects. This gRNA was integrated into the pRGEB32 binary vector at the BsaI restriction sites. In this construct, both the codon-optimized Cas9 and the gRNA scaffold are driven by the rice ubiquitin promoter to ensure robust expression. Following ligation, the recombinant plasmids were transformed into E. coli TOP10. We confirmed successful transformants through a combination of colony PCR and restriction enzyme digestion, with the final gRNA orientation and sequence integrity being verified via Sanger sequencing

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