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

Area of Science:

• Plant biotechnology
• Genomics
• Agricultural science

Background:

• Prolonged expression of CRISPR-Cas9 components in gene-edited plants can hinder trait analysis and increase off-target mutations.
• Controlling gene editing component expression is crucial for improving biosafety and efficiency in crop improvement.

Purpose of the Study:

• To identify and characterize novel callus-specific promoters in peanut (Arachis hypogaea L.).
• To evaluate the efficacy of these promoters in driving Cas9-mediated cytosine base editing specifically in peanut callus.
• To provide tools for enhancing the biosafety of genome editing in peanut breeding.

Main Methods:

• Mining RNA sequencing datasets to identify six candidate callus-specific genes in peanut.
• Validating gene expression profiles using quantitative reverse transcriptase PCR.
• Cloning and assessing the activity of five identified promoters using beta-glucuronidase (GUS) staining.
• Evaluating the ability of promoters to drive cytosine base editing using a deaminase-nCas9 fusion protein.

Main Results:

• Five peanut promoters (P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-I20Q6X, P_Ah-ELJ55T, and P_Ah-N9CMH4) were confirmed to be functional in peanut callus.
• All five promoters successfully drove precise base substitutions in peanut via cytosine base editing.
• Four promoters (P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4) demonstrated editing efficiencies comparable to or exceeding the cauliflower mosaic virus 35S promoter.

Conclusions:

• The identified callus-specific promoters offer a valuable tool for precise and efficient genome editing in peanut.
• Restricting Cas9 expression to the callus stage significantly improves the biosafety of CRISPR applications in peanut.
• These promoters can be instrumental in accelerating peanut breeding programs through enhanced genome editing strategies.