RNA-Seq Identification of Peanut Callus-Specific Promoters and Evaluation of Base-Editing Efficiency

Prolonged expression of gene-editing components in CRISPR-modified plants can interfere with phenotypic analysis of target traits, increase the risk of off-target mutations, and lead to unnecessary metabolic burden. To mitigate these issues in peanut (Arachis hypogaea L.), callus-specific promoters were screened to restrict Cas9 expression to the callus stage, minimizing its activity in regenerated plants. In this study, six callus-specific genes in peanut were identified by mining RNA sequencing datasets and validating their expression profiles using quantitative reverse transcriptase PCR. The promoters of Arahy.H0FE8D, Arahy.WT3AEF, Arahy.I20Q6X, Arahy.ELJ55T, and Arahy.N9CMH4 were cloned and assessed for their expression activity. Beta-glucuronidase (GUS) histochemical staining confirmed that all five promoters were functional in peanut callus. Further investigation revealed their ability to drive cytosine base editing via a deaminase-nCas9 fusion protein, with all promoters successfully inducing precise base substitutions in peanut. Notably, P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4 exhibited comparable or higher editing efficiencies than the commonly used cauliflower mosaic virus 35S promoter. These findings provide valuable tools for improving the biosafety of CRISPR-based genome editing in peanut breeding programs.