Zhang, Y., Massel, K., Godwin, I. D. & Gao, C. Applications and potential of genome editing in crop improvement. Genome Biol. 19, 210 (2018).
Google Scholar
Ahmar, S., Hensel, G. & Gruszka, D. CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals: current status, improvements, and perspectives. Biotechnol. Adv. 69, 108248 (2023).
Google Scholar
Gilbertson, L., Puchta, H. & Slotkin, R. K. The future of genome editing in plants. Nat. Plants 11, 680–685 (2025).
Google Scholar
Gao, Y. & Zhao, Y. Epigenetic suppression of T-DNA insertion mutants in Arabidopsis. Mol. Plant 6, 539–545 (2013).
Google Scholar
Schubert, D. et al. Silencing in Arabidopsis T-DNA transformants: The predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell 16, 2561–2572 (2004).
Google Scholar
Daxinger, L. et al. Unexpected silencing effects from T-DNA tags in Arabidopsis. Trends Plant Sci. 13, 4–6 (2008).
Google Scholar
Kirchhoff, J. et al. Gene expression variability between randomly and targeted transgene integration events in tobacco suspension cell lines. Plant Biotechnol. Rep. 14, 451–458 (2020).
Google Scholar
Olhoft, P. M., Flagel, L. E. & Somers, D. A. T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol. J. 2, 289–300 (2004).
Google Scholar
Thomson, G., Dickinson, L. & Jacob, Y. Genomic consequences associated with Agrobacterium-mediated transformation of plants. Plant J. 117, 342–363 (2024).
Google Scholar
Petolino, J. F. & Kumar, S. Transgenic trait deployment using designed nucleases. Plant Biotechnol. J. 14, 503–509 (2016).
Google Scholar
Dong, O. X. & Ronald, P. C. Targeted DNA insertion in plants. Proc. Natl Acad. Sci. USA 118, e2004834117 (2021).
Google Scholar
Tuncel, A. et al. Genome-edited foods. Nat. Rev. Bioeng. 1, 799–816 (2023).
Google Scholar
Přibylová, A. & Fischer, L. How to use CRISPR/Cas9 in plants: from target site selection to DNA repair. J. Exp. Bot. 75, 5325–5343 (2024).
Google Scholar
Schreiber, T. et al. Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases. Mol. Plant 17, 824–837 (2024).
Google Scholar
Chen, J., Li, S., He, Y., Li, J. & Xia, L. An update on precision genome editing by homology-directed repair in plants. Plant Physiol. 188, 1780–1794 (2022).
Google Scholar
Zong, Y. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40, 1394–1402 (2022).
Google Scholar
Pandey, S. et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat. Biomed. Eng. 9, 22–39 (2025).
Google Scholar
Puchta, H., Dujon, B. & Hohn, B. Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res. 21, 5034–5040 (1993).
Google Scholar
Puchta, H. & Fauser, F. Gene targeting in plants: 25 years later. Int. J. Dev. Biol. 57, 629–637 (2013).
Google Scholar
Van Vu, T. et al. Challenges and perspectives in homology-directed gene targeting in monocot plants. Rice 12, 95 (2019).
Google Scholar
Liu, P. et al. Transposase-assisted target-site integration for efficient plant genome engineering. Nature 631, 593–600 (2024).
Google Scholar
Li, R. et al. Method for biolistic site-specific integration in plants catalyzed by Bxb1 integrase. Methods Mol. Biol. 1469, 15–30 (2016).
Google Scholar
McCormick, S. Recombinases and rhizogenes for easy gene stacking. Plant J. 95, 571–572 (2018).
Google Scholar
Sun, C. et al. Iterative recombinase technologies for efficient and precise genome engineering across kilobase to megabase scales. Cell 188, 4693–4710.e4615 (2025).
Google Scholar
Dong, O. X. et al. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. Nat. Commun. 11, 1178 (2020).
Google Scholar
Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42, 316–327 (2024).
Google Scholar
Shang, L. et al. A complete assembly of the rice Nipponbare reference genome. Mol. Plant 16, 1232–1236 (2023).
Google Scholar
Tulpová, Z. et al. Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi-omics approach. Plant Genome 15, e20191 (2022).
Google Scholar
Chen, J. et al. A complete telomere-to-telomere assembly of the maize genome. Nat. Genet. 55, 1221–1231 (2023).
Google Scholar
Sims, J., Sestini, G., Elgert, C., von Haeseler, A. & Schlogelhofer, P. Sequencing of the Arabidopsis NOR2 reveals its distinct organization and tissue-specific rRNA ribosomal variants. Nat. Commun. 12, 387 (2021).
Google Scholar
Chen, W. et al. The complete genome assembly of Nicotiana benthamiana reveals the genetic and epigenetic landscape of centromeres. Nat. Plants 10, 1928–1943 (2024).
Google Scholar
Zupunski, V., Gubensek, F. & Kordis, D. Evolutionary dynamics and evolutionary history in the RTE clade of non-LTR retrotransposons. Mol. Biol. Evol. 18, 1849–1863 (2001).
Google Scholar
Fedoroff, N. V. Presidential address. Transposable elements, epigenetics, and genome evolution. Science 338, 758–767 (2012).
Google Scholar
Wells, J. N. & Feschotte, C. A field guide to eukaryotic transposable elements. Annu. Rev. Genet. 54, 539–561 (2020).
Google Scholar
Malik, H. S., Burke, W. D. & Eickbush, T. H. The age and evolution of non-LTR retrotransposable elements. Mol. Biol. Evol. 16, 793–805 (1999).
Google Scholar
Zhang, X. et al. Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci. Nat. Biotechnol. 43, 42–51 (2025).
Google Scholar
Thawani, A., Florez Ariza, A. J., Nogales, E. & Collins, K. Template and target-site recognition by human LINE-1 in retrotransposition. Nature 626, 186–193 (2024).
Google Scholar
Han, J. S. Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions. Mob DNA 1, 15 (2010).
Google Scholar
Wilkinson, M. E., Frangieh, C. J., Macrae, R. K. & Zhang, F. Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription. Science 380, 301–308 (2023).
Google Scholar
Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605 (1993).
Google Scholar
Eickbush, D. G., Luan, D. D. & Eickbush, T. H. Integration of Bombyx mori R2 sequences into the 28S ribosomal RNA genes of Drosophila melanogaster. Mol. Cell. Biol. 20, 213–223 (2000).
Google Scholar
Christensen, S. M., Ye, J. & Eickbush, T. H. RNA from the 5′ end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site. Proc. Natl Acad. Sci. 103, 17602–17607 (2006).
Google Scholar
Eickbush, T. H. & Eickbush, D. G. Integration, regulation, and long-term stability of R2 retrotransposons. Microbiol. Spectr. 3, Mdna3-0011-2014 (2015).
Google Scholar
Palm, S. M., Horton, C. A., Zhang, X. & Collins, K. Structure and sequence at an RNA template 5′ end influence insertion of transgenes by an R2 retrotransposon protein. RNA 30, 1227–1245 (2024).
Google Scholar
Xiong, Y. & Eickbush, T. H. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons. Mol. Cell. Biol. 8, 114–123 (1988).
Google Scholar
Kojima, K. K. & Fujiwara, H. Long-term inheritance of the 28S rDNA-specific retrotransposon R2. Mol. Biol. Evol.22, 2157–2165 (2005).
Google Scholar
Burke, W. D., Muller, F. & Eickbush, T. H. R4, a non-LTR retrotransposon specific to the large subunit rRNA genes of nematodes. Nucleic Acids Res. 23, 4628–4634 (1995).
Google Scholar
Burke, W. D., Singh, D. & Eickbush, T. H. R5 retrotransposons insert into a family of infrequently transcribed 28S rRNA genes of planaria. Mol. Biol. Evol. 20, 1260–1270 (2003).
Google Scholar
Kojima, K. K. & Fujiwara, H. Evolution of target specificity in R1 clade non-LTR retrotransposons. Mol. Biol. Evol. 20, 351–361 (2003).
Google Scholar
Gladyshev, E. A. & Arkhipova, I. R. Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion. Gene 448, 145–150 (2009).
Google Scholar
Besansky, N. J., Paskewitz, S. M., Hamm, D. M. & Collins, F. H. Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae. Mol. Cell. Biol. 12, 5102–5110 (1992).
Google Scholar
Kojima, K. K., Kuma, K., Toh, H. & Fujiwara, H. Identification of rDNA-specific non-LTR retrotransposons in Cnidaria. Mol. Biol. Evol. 23, 1984–1993 (2006).
Google Scholar
Burke, W. D., Malik, H. S., Jones, J. P. & Eickbush, T. H. The domain structure and retrotransposition mechanism of R2 elements are conserved throughout arthropods. Mol. Biol. Evol. 16, 502–511 (1999).
Google Scholar
Kuroki-Kami, A. et al. Targeted gene knockin in zebrafish using the 28S rDNA-specific non-LTR-retrotransposon R2Ol. Mob. DNA 10, 23 (2019).
Google Scholar
Yang, J., Malik, H. S. & Eickbush, T. H. Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements. Proc. Natl Acad. Sci. USA 96, 7847–7852 (1999).
Google Scholar
Deng, P. et al. Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon. Cell 186, 2865–2879 e2820 (2023).
Google Scholar
Thawani, A. et al. Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second-strand nicking. Sci. Adv. 11, eadu5533 (2025).
Google Scholar
Tang, S. & Sternberg, S. H. Genome editing with retroelements. Science 382, 370–371 (2023).
Google Scholar
Luo, S., Chen, Q., Chen, Y. & Li, W. Biology and utilization of R2 retrotransposons. RNA Biol. 22, 1–8 (2025).
Google Scholar
Chen, Y. et al. All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons. Cell 187, 4674–4689.e4618 (2024).
Google Scholar
Edmonds, K. K. et al. Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons. Nat. Commun. 16, 6079 (2025).
Google Scholar
Fell, C. W. et al. Reprogramming site-specific retrotransposon activity to new DNA sites. Nature 642, 1080–1089 (2025).
Google Scholar
Kawahara, Y. et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N Y) 6, 4 (2013).
Google Scholar
Hamilton, J. P., Li, C. & Buell, C. R. The rice genome annotation project: an updated database for mining the rice genome. Nucleic Acids Res. 53, D1614–d1622 (2025).
Google Scholar
Butt, H. et al. Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide RNA molecule. Front. Plant Sci. 8, 1441 (2017).
Google Scholar
Ali, Z. et al. Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice. Commun. Biol. 3, 44 (2020).
Google Scholar
Molla, K. A., Sretenovic, S., Bansal, K. C. & Qi, Y. Precise plant genome editing using base editors and prime editors. Nat. Plants 7, 1166–1187 (2021).
Google Scholar
Moran, J. V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).
Google Scholar
Ostertag, E. M., Prak, E. T., DeBerardinis, R. J., Moran, J. V. & Kazazian, H. H. Jr. Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Res. 28, 1418–1423 (2000).
Google Scholar
McIntyre, J. J. R., Horton, C. A. & Collins, K. Different DNA repair pathways support intact or truncated insertions by R2 retrotransposon protein. Science 391, eadz3121 (2026).
Google Scholar
De Paepe, A. et al. High frequency of single-copy T-DNA transformants produced by floral dip in CRE-expressing Arabidopsis plants. Plant J. 59, 517–527 (2009).
Google Scholar
Ali, Z. et al. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol. Plant 8, 1288–1291 (2015).
Google Scholar
Liu, Q., Zhao, C., Sun, K., Deng, Y. & Li, Z. Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes. Mol. Plant 16, 616–631 (2023).
Google Scholar
Muchenje, K. T. et al. Optimized R2 retroelement complexes enable precise and efficient DNA insertion into plant genomes. Preprint at bioRxiv https://doi.org/10.1101/2025.08.22.671877 (2025).
Gibbons, J. G., Branco, A. T., Yu, S. & Lemos, B. Ribosomal DNA copy number is coupled with gene expression variation and mitochondrial abundance in humans. Nat. Commun. 5, 4850 (2014).
Google Scholar
Wang, Z. P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).
Google Scholar
Hiei, Y. & Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 3, 824–834 (2008).
Google Scholar
Ali, Z., Butt, H., Alghamdi, R., Ramirez, J. L. M. & Mahfouz, M. Efficient site-specific gene addition using R2 retrotransposons in tobacco and rice [datasets] (NCBI Sequence Read Archive, 2026).