Development of a prime editor with improved editing efficiency in Arabidopsis
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Prime editing is widely used in many organisms to introducesite-specific sequence modifications such as base substitutions,insertions, and deletions in genomic DNA without generatingdouble-strand breaks. Despite wide-ranging applications ofprime editing, prime editors (PEs) have low editing efficiencies,especially in dicot plants. Therefore, PEs are barelyused for genome engineering in dicot plant species. Here,based on previous approaches used to improve prime editingefficiency, we generated different combinations of PE componentsand prime editing guide RNAs (pegRNAs) and examinedtheir prime editing efficiencies in Arabidopsis thaliana protoplastsas a dicot model system. We found that v4e2, in whichPE was fused to viral nucleocapsid (NC) protein, RNase HdeletedM-MLV RT, and a dominant negative version of humanmutL homolog 1 (hMLH1dn), showed the highest prime editingefficiency in Arabidopsis protoplasts when it was co-transfectedwith dual enhanced pegRNA. Our results suggest that the v4e2 PEsystem could be used for efficient prime editing in dicot plants.
Abstract
Prime editing is widely used in many organisms to introducesite-specific sequence modifications such as base substitutions,insertions, and deletions in genomic DNA without generatingdouble-strand breaks. Despite wide-ranging applications ofprime editing, prime editors (PEs) have low editing efficiencies,especially in dicot plants. Therefore, PEs are barelyused for genome engineering in dicot plant species. Here,based on previous approaches used to improve prime editingefficiency, we generated different combinations of PE componentsand prime editing guide RNAs (pegRNAs) and examinedtheir prime editing efficiencies in Arabidopsis thaliana protoplastsas a dicot model system. We found that v4e2, in whichPE was fused to viral nucleocapsid (NC) protein, RNase HdeletedM-MLV RT, and a dominant negative version of humanmutL homolog 1 (hMLH1dn), showed the highest prime editingefficiency in Arabidopsis protoplasts when it was co-transfectedwith dual enhanced pegRNA. Our results suggest that the v4e2 PEsystem could be used for efficient prime editing in dicot plants.