Optimized R2 Retroelement Complexes Enable Precise and Efficient DNA Insertion into Plant Genomes

Precise, targeted insertion of multi-kilobase DNA sequences is critical for studying gene function, ensuring robust transgene expression, and stacking traits in crops, but remains difficult to achieve. Existing targeted insertion methods in plants relying on programmable nucleases are inefficient and can generate unwanted mutations. Newer technologies based on prime editors, transposases, and site-specific recombinases extend capabilities but remain constrained with low efficiencies, off-target integration, silencing, or limited cargo DNA size. R2 non-long terminal repeat (non-LTR) retrotransposons integrate via target-primed reverse transcription specifically targeting the 25S ribosomal DNA multicopy site and enabling double-strand-break-free installation of gene-sized DNA sequences. Here, we adapted the avian Taeniopygia guttata R2 protein (R2Tg) for targeted DNA insertion into plant genomes and defined design rules for efficient integration and functional protein expression from the 25S rDNA locus. We engineered R2Tg expression cassettes and RNA payloads carrying an intron-disrupted mCherry retrotransposition reporter with length-optimized rDNA homology arms. We also optimized the R2 editor delivery format and incubation conditions. In Nicotiana benthamiana leaves, Arabidopsis thaliana protoplasts, and Solanum lycopersicum seedlings, the optimized R2Tg editor system achieved targeted insertion with efficiencies up to 24% for 2-5kb size payloads, which were carefully validated at the molecular level. This work establishes a compact, ribonucleoprotein-based platform for targeted DNA insertion into plant genomes, leveraging a multicopy genomic safe harbor site to enable efficient multi-kilobase precise gene addition.