Precise deletion, replacement, and inversion of large DNA fragments in plants using dual prime editing

Precise manipulation of genome structural variations (SVs) holds significant potential for plant trait improvement and biological research. Here, we present a genome-editing approach, dual prime editing (DualPE), that efficiently facilitates precise deletion, replacement, and inversion of large DNA fragments in plants. DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in dicot Nicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome editing for crop improvement.