茄科作物高效双碱基编辑新工具的开发与应用OA

This study seeks to develop a highly efficient dual-base editing tool for solanaceous crops to address the challenges of poor compatibility and low editing efficiency associated with existing base editing systems in these plants.The research involved fusing the nickase Cas9 enzyme(nCas9),which has partially lost its nuclease function,with components such as the highly efficient adenine deaminase ABE8e,the optimized cytosine deaminase sdd7-mini,and the uracil glycosylation inhibitor UGI.Systematic optimization was conducted based on the promoter preferences and co-don usage frequencies specific to solanaceous crops.Ultimately,the dual-base editor TPDBE-S was constructed.In tran-sient transformation experiments using tomato and eggplant protoplasts,this editor exhibited exceptionally high editing ef-ficiency.In the stable transformation system of tobacco,targeting the PDS gene verification revealed that four out of five positive transgenic families exhibited pronounced albinism phenotypes,thereby confirming the stable editing capability.The editing window of this editor aligns closely with the characteristics of ABE8e and sdd7-mini.Furthermore,optimizing codon preference and promoter activity in Solanaceae crops significantly enhances editing efficiency and adaptability.The construction of TPDBE-S employs a modular design.The multi-target expression cassette,based on the tRNA self-cleaving mechanism,can be rapidly assembled through one-step gene synthesis,which is both cost-effective and straight-forward.This tool addresses the gap in double-base editing specifically for Solanaceous crops.