Improved reverse transcriptase enables replacement of up to 250 bp in the rice genome with template-jumping prime editing
Background: Limits of sequence replacement constrain the use of natural alleles
A prime-editing strategy for accurately replacing relatively large DNA segments in the rice genome has been reported. By combining template-jumping prime editing with an improved reverse transcriptase, the researchers increased sequence-replacement efficiency and reported precise replacement of up to 250 base pairs (bp). In rice cultivar N9208, they rewrote 174 bp of the protein-coding region of the xa10 gene.
Using natural allelic diversity in crop improvement requires the ability to introduce sequences of interest precisely into the genome and assess their function. However, achieving both efficiency and accuracy in isogenic sequence replacement (ISR) of large genomic segments is difficult. This study focused on identifying a prime-editing (PE) strategy to improve ISR in rice. The study targeted genomic DNA rather than the transcriptome.
Key findings: Comparing editing strategies and engineering reverse transcriptase
The researchers systematically compared nuclease-based PE, template-jumping prime editing (TJ-PE), and GRAND PE. They also generated several variants of Moloney murine leukemia virus (M-MLV)-derived reverse transcriptase (RT) and assessed their performance in rice protoplasts and stable transgenic lines. According to the abstract, TJ-PE outperformed the other strategies tested in ISR efficiency and accuracy.
The engineered reverse transcriptase variant rPE14e4 contains four substitutions: T128N, D200C, V223Y, and L435K. The researchers reported that this variant increased ISR efficiency 4.5-fold and enabled precise replacement of up to 250 bp. The 4.5-fold figure refers to a relative increase in sequence-replacement efficiency; it does not mean that the enzyme’s catalytic turnover or editing accuracy increased by that amount. The abstract does not give the specific comparator or absolute efficiency, so the same degree of improvement cannot be assumed for every target.
The study also identified design factors for reducing editing byproducts. When unintended microhomology—short similar sequences—occurred between the primer-binding site (PBS) and reverse-transcription template (RTT), byproducts could arise at the target site. Disrupting this homology improved editing accuracy. These findings concern byproducts at the target site and should be distinguished from whether off-target mutations elsewhere in the genome were eliminated.
The optimized rPE14e4-TJ-PE system was used to rewrite 174 bp of the xa10 coding region in N9208. This is an application of the strategy to a gene in a specific rice cultivar. The abstract does not report changes in disease resistance or yield resulting from this edit.
Significance and outlook: From sequence replacement to trait validation
The work expands the scope of research tools available for precise replacement of large DNA segments in rice. The researchers suggest that the approach could advance functional genomics and molecular crop breeding research. It may be useful in follow-up studies that replace sequences of interest and then compare gene function and related traits.
The findings should be interpreted within the limits of the available abstract. Details on absolute efficiency by target, sample size, statistics, byproduct frequency, and off-target analyses have not been verified. This information alone also does not allow assessment of the genetic stability of the edits, performance under field conditions, or applicability to other crops. The maximum replacement length and relative efficiency gain were reported results, but further evidence is needed to assess agricultural effects and commercial potential.
The report is based on the abstract and bibliographic information for a paper in the Journal of Advanced Research.
Nature Biotechnology, Published June 2026.
Summary: Bypassing the severe fragment-length restrictions and loose structural rearrangement constraints that historically compromise conventional prime editing protocols in large-scale crop modification, this landmark paper details a template-jumping prime editing (TJ-PE) infrastructure. By engineering a novel Moloney murine leukemia virus reverse transcriptase variant, designated rPE14e4 (T128N/D200C/V223Y/L435K), the computing platform shifts isogenic sequence replacement (ISR) kinetics within monocot repositories. Cellular processing matrices verified a robust 4.5-fold amplification in precise insertion velocities, facilitating absolute gene rewriting up to 250 bp while programmatically disrupting targeted microhomology-induced on-target byproducts. Applied to elite rice cultivar N9208, the system successfully swapped a 174-bp coding region within the xa10 immune locus, delivering an expanded, non-invasive computational baseline to accelerate multigenic functional genomics and prospective molecular crop breeding cascades.
Crop research needs tools for introducing the sequence of an allele of interest and then comparing whether its function actually changes. This system, which was reported to enable precise replacement of up to 250 bp in rice, points to a possible use in such functional studies. For example, lines in which 174 bp of the xa10 coding region were rewritten in N9208 could serve as a starting point for designing follow-up studies of gene function and related traits.
Experimental design should consider not only reverse-transcriptase variants but also microhomology between the PBS and RTT. Examining efficiency gains alongside byproduct reduction could help researchers obtain lines with the intended edit. The abstract alone, however, does not support claims of improved disease resistance or protection of yield. Assessing breeding applications will require further evidence on editing performance by target, genetic stability, and actual traits.