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

Precise, targeted insertion of multi-kilobase DNA sequences into plant genomes is critical for studying gene function, ensuring robust transgene expression, and stacking traits in crops, but remains challenging. 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 DNA payload size. R2 non-long terminal repeat (non-LTR) retrotransposons integrate via target-primed reverse transcription (TPRT) specifically targeting the 25S ribosomal DNA multicopy site and enabling double-strand-break-free installation of gene-sized DNA sequences. We adapted the avian Taeniopygia guttata R2 protein (R2Tg) for targeted DNA insertion into plant genomes through engineering of R2Tg expression cassettes and RNA payloads carrying intron-disrupted mCherry and RUBY retrotransposition reporters with length-optimized rDNA homology arms. These efforts, together with optimized construct delivery formats and incubation temperatures, define R2 editor design rules enabling efficient DNA integration and functional protein expression from the 25S rDNA locus. In Nicotiana benthamiana leaves, Arabidopsis thaliana protoplasts, and Solanum lycopersicum seedlings, the optimized R2Tg editor system achieved targeted insertion with efficiencies up to 24% payloads ranging in size from 2kb to 5kb. This work establishes a compact R2Tg ribonucleoprotein platform for targeted DNA insertion into plant genomes, targeting a multicopy genomic safe harbor site to enable efficient multi-kilobase gene addition