Expanding the Scope of Prime Editing in Rice from Hundreds of Base Substitutions to Thousands of Base Deletions
Background
Crop traits are influenced not only by single nucleotide variations but also by sequence differences spanning tens to hundreds of nucleotides, including regulatory regions and protein domains. Replacing entire genomic regions of elite varieties with desired alleles requires large-scale homologous replacement, but achieving both efficiency and precision has been challenging in plants.
Conventional CRISPR-Cas9-based homology-directed repair involves cleaving DNA double strands and supplying an external template. However, plant cells tend to prioritize non-homologous end joining, leading to insertion/deletion byproducts, and editing efficiency is significantly affected during tissue culture and plant regeneration. Prime editing (PE) utilizes Cas9 nickase, reverse transcriptase, and prime editing guide RNA (pegRNA) to introduce desired information into the target site without double-strand cleavage. However, conventional PE is typically limited to short substitutions and insertions/deletions, with significant constraints on replacing sequences longer than 100 base pairs.
Key Findings
The researchers expanded template-jumping prime editing (TJ-PE), a strategy reported for large DNA insertions, to homologous replacement in rice. TJ-PE is designed to allow the reverse transcriptase to continuously read the editing template, connecting the newly synthesized DNA to the opposite end of the target locus. This reduces the structural burden of conventional PE, which requires accommodating the entire long sequence within the reverse transcriptase template of a single pegRNA.
In the rice genome, the researchers replaced DNA fragments of various lengths, from tens to hundreds of base pairs, with homologous sequences of the same length. The longest replaced region was 340 base pairs. The key finding is that they successfully removed the original genomic fragment and replaced it with a designed sequence of the same length, rather than simply adding nucleotides. This demonstrates the potential to move multiple variants present in natural alleles or consecutive functional motifs in a region-by-region manner.
The application of TJ-PE is not limited to substitutions. The researchers precisely deleted genomic fragments of 944 to 2,024 base pairs at defined locations. Even under conditions where approximately 2,000 base pairs were removed, the highest efficiency recorded was 34.6%. As a result, it is now possible to perform both precise replacement of large fragments and kilobase-scale deletions using the same editing system.
Significance and Outlook
This result expands the editing scale of PE in rice from single nucleotides or short sequences to the level of gene functional regions. Alleles associated with disease resistance, environmental stress adaptation, yield, and quality often contain multiple variants clustered together. If TJ-PE can be reliably applied, it will be possible to reproduce these useful sequence clusters at once, rather than editing each variant individually. It can also be used to remove long regulatory sequences to alter gene expression or to replace regions encoding specific protein domains.
However, it is difficult to assume that the maximum efficiency shown will be reproduced in all targets and varieties. Performance may vary depending on guide RNA structure, target flanking sequences, and chromatin accessibility, and the ratio of accurate substitutions to partial edits and off-target byproducts should be thoroughly verified. The stability of edited traits across generations, the stability of agronomic traits, and the evaluation of off-target variations at the whole-genome level are also important tasks to be confirmed before commercial breeding.
It remains to be seen whether the technology can be extended to crops with larger genomes or higher ploidy levels, such as wheat and maize. This will be a key focus of future research.
Homologous replacement of genomic sequences with large DNA fragments (> 100 bp) holds great potential for crop breeding, yet an efficient method to achieve such edits is lacking in plants. Here, in rice, we developed template-jumping prime editing (TJ-PE), a recently reported PE strategy for large targeted insertion, as an efficient tool for homologous replacement with DNA fragments ranging from dozens to hundreds of base pairs, and using TJ-PE, we replaced genomic fragments of up to 340 bp with homologous fragments of the same length. In addition, our TJ-PE tool also enabled precise deletion of 944- to 2024-bp fragments in rice, with efficiencies of up to 34.6% for c. 2000-bp precise deletions. Collectively, this study expands the editing scope of PE in rice and establishes TJ-PE as a generalist tool for precise deletion and replacement of large DNA fragments.
Seed companies can consider strategies to move beneficial alleles of elite varieties, associated with disease resistance or quality, from one variety to another by transferring hundreds of base pair regions, rather than recreating them base by base. For example, this could involve replacing multiple functional motifs of a promoter at once or precisely deleting an unfavorable regulatory region of approximately 2 kilobases to regulate gene expression. The fact that it precisely modifies the existing genome without randomly inserting foreign genes is also advantageous for crop development.
However, in actual breeding, it is necessary to select and analyze the progeny after the editing reagents have been removed, and to confirm the absence of unintended variations and the stability of traits through whole-genome analysis and multiple generations of field trials. Given that TJ-PE efficiency is likely to vary depending on the variety and target, the development of high-efficiency guides and the standardization of plant regeneration processes will be key factors in determining the speed of industrial application.