Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system

Area of Science:

• Plant biotechnology
• Gene editing technologies
• Crop improvement

Background:

• CRISPR/Cas-base editing offers precise nucleotide conversion.
• Developing efficient base editing systems for allotetraploid crops like Brassica napus is crucial.

Purpose of the Study:

• To establish and evaluate the A3A-PBE base-editing system in Brassica napus.
• To assess editing efficiency, inheritance, and potential applications of base-edited genes.

Main Methods:

• Developed the A3A-PBE system using human A3A cytidine deaminase, Cas9 nickase, and uracil glycosylase inhibitor.
• Designed sgRNAs targeting ALS, RGA, and IAA7 genes.
• Analyzed base-editing efficiency, editing window, inheritance, and off-target mutations via sequencing.

Main Results:

• Achieved >20% base-editing efficiency for ALS, RGA, and IAA7 genes.
• Observed an editing window from C1 to C10 of the PAM sequence.
• Base-edited ALS plants showed herbicide resistance; RGA/IAA7 plants had reduced height, with all edits being heritable.

Conclusions:

• The A3A-PBE system is effective for C-to-T substitutions in oilseed rape with a broad editing window.
• Base-edited mutants have potential applications in herbicide resistance and improved plant architecture for mechanical harvesting.