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Author: Yangcan Chen

  • Discovery and engineering of avian R2 retrotransposons for all-RNA-mediated targeted DNA integration in human cells

    Discovery and engineering of avian R2 retrotransposons for all-RNA-mediated targeted DNA integration in human cells

    Data availability

    The sequencing data involved in this research have been submitted to the Genome Sequence Archive for Human, a platform under the Beijing Institute of Genomics, Chinese Academy of Sciences (available at https://ngdc.cncb.ac.cn/gsa-human/). As of the paper’s publication date, the data will be open to the public, and the accession number for retrieval is HRA013312 (ref. 48). Source data are provided with this paper.

    Code Availability

    Analytic code for the sequencing data is publicly available at GitHub at https://github.com/YanpingHu/avian_R2 (ref. 49).

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    Acknowledgements

    We would like to thank J. Sun for guidance on T cell manipulation. We also thank the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, for providing the primary HHFs used in this study.

    Funding

    This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA0510400 and XDC0200000 to W.L. and H.W.; the National Natural Science Foundation of China (32225030 and 82488301 to W.L.; 32501337 to Y.C.; 32425035 and T2521004 to H.W.); the National Key Research and Development Program (2024YFA0917300 to W.L. and N.T.; 2019YFA0110800 to W.L.); the Beijing Natural Science Foundation (Z230011 to W.L.); Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences (2023IOZ0204, 2024IOZ0202, 2025IOZ03); the Agriculture Science and Technology Major Project (W.L. and H.W.); the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (GZB20240739 to Y.H.) and the New Cornerstone Science Foundation through the XPLORER PRIZE.

    Author information

    Author notes

    1. These authors contributed equally: Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang.

    Authors and Affiliations

    1. State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

      Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang, Hanyu Bai, Xinge Wang, Changxian Peng, Qicheng Chen, Jiahui Xiao, Yu Zou, Peng Wang, Rui Niu, Jin Zhang, Yulong Zhao, Chen Liang, Yunlong Wang, Yiyun Li, Junjie Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li

    2. University of Chinese Academy of Sciences, Beijing, China

      Yangcan Chen, Shengqiu Luo, Bangwei Mao, Yuze Wang, Hanyu Bai, Changxian Peng, Qicheng Chen, Jiahui Xiao, Yu Zou, Rui Niu, Jin Zhang, Yulong Zhao, Chen Liang, Yunlong Wang, Yiyun Li, Junjie Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li

    3. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China

      Yangcan Chen, Yanping Hu, Na Tang, Chenxin Wang, Xinge Wang, Peng Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li

    Authors

    1. Yangcan Chen
    2. Yanping Hu
    3. Shengqiu Luo
    4. Bangwei Mao
    5. Na Tang
    6. Chenxin Wang
    7. Yuze Wang
    8. Hanyu Bai
    9. Xinge Wang
    10. Changxian Peng
    11. Qicheng Chen
    12. Jiahui Xiao
    13. Yu Zou
    14. Peng Wang
    15. Rui Niu
    16. Jin Zhang
    17. Yulong Zhao
    18. Chen Liang
    19. Yunlong Wang
    20. Yiyun Li
    21. Junjie Wang
    22. Ng Shyh-Chang
    23. Tuo Wei
    24. Haoyi Wang
    25. Qi Zhou
    26. Wei Li

    Contributions

    W.L., Q.Z. and Y.C. conceived the project and designed the experiments. Y.C., Y.H., S.L., B.M., N.T., C.W., Yuze Wang, H.B., X.W., C.P., Q.C., J.X., Y. Zou, P.W., R.N., J.Z., Y. Zhao, C.L., Yunlong Wang, Y.L. and J.W. performed the experiments. W.L., Q.Z., Y.C., Y.H. and S.L. analyzed the data. W.L., Q.Z., Y.C. and H.W. supervised the research. W.L., Q.Z., Y.C., Y.H., S.L., N.S.-C., T.W. and H.W. wrote the paper, assisted by the other authors.

    Corresponding authors

    Correspondence to Yangcan Chen, Haoyi Wang, Qi Zhou or Wei Li.

    Ethics declarations

    Competing interests

    W.L., Q.Z., Y.C., Y.H., S.L., Q.C., X.W. and B.M. have submitted patent applications related to this paper. The other authors declare no competing interests.

    Peer review

    Peer review information

    Nature Biotechnology thanks Lei Qi, Max Wilkinson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

    Additional information

    Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

    Extended data

    Extended Data Fig. 1 Workflow for mining new R2 retrotransposons in avian genomes.

    a, Computational pipeline for identifying avian R2 retrotransposons. b, Distribution of newly discovered R2 elements across orders of analyzed avian genomes. c, Left: distribution of downloaded avian genomes across different orders; Right: distribution of avian genomes containing full-length R2 elements across different orders.

    Extended Data Fig. 2 Systematic characterization of Region-1 and Region-2 in the R2Tg protein.

    a, Droplet digital PCR (ddPCR) analysis demonstrates that Region-1 truncation markedly impairs the molecular integration efficiency of R2Tg, and this defect can be rescued by HMGN1 fusion. Cp, copies. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test. b, Effect of Region-1 truncation length on R2-mediated gene integration efficiency in HEK293T cells, coupled with donoropt. c, Identification of substituting domains capable for rescuing Region-1 function in HEK293T cells, coupled with donoropt. NLS1/2, Nuclear Localization Signal 1/217; NBD, nucleosome-binding domain; CHUD, chromatin unfolding domain. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test. d, Trans-expression of Region-1 fails to rescue R2-mediated gene integration efficiency in HEK293T cells, coupled with donoropt. Unpaired, two-tailed Student’s t tests. e, Impact of region-2 truncation on R2-mediated gene integration activity in HEK293T cells, coupled with donoropt. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test. f, Statistical analysis of Region-1 length across 165 avian R2 retrotransposon proteins. g, Statistical analysis of length of distinct subregions (Spacer, NTE-2 and partial NTE-1) in Region-2 across 165 avian R2 retrotransposon proteins. h, Evaluating the activity of R2Tg protein with removed nuclear localization signal or repositioned Flag tag in all-RNA experimental systems in HEK293T cells, coupled with donoropt. Unless otherwise stated, data are presented as mean ± standard deviation (SD) from three biological replicates.

    Extended Data Fig. 3 Investigation of the effects of donor RHA length and polyA length on R2-mediated gene integration efficiency.

    a, Impact of donor RNA with varying right homology arm (RHA) and polyA lengths on integration efficiency in HEK293T cells. US, unrelated vector sequence. b, Impact of donor RNA with 4 nt RHA and various polyA lengths in HEK293T cells. c, Integration efficiency of R2Tgv1.1 with donor RNAs containing different nucleotide components after a 4 bp RHA in HEK293T cells. Unless otherwise stated, data are presented as mean ± standard deviation (SD) from three biological replicates.

    Extended Data Fig. 4 Investigation of the effects of 5’ UTR elements on R2-mediated gene integration efficiency.

    a, Statistical analysis of J1/2 region length across 165 avian R2 5’ ribozymes. b, Predicted secondary structure of the J1/2 region in 165 avian R2 5’ ribozymes, inferred via covariation modeling. c, Statistical analysis of the length of non-ribozyme part in 165 R2 5’ UTRs. d, Predicted secondary structure of the non-ribozyme part in avian R2 5’ UTRs from Aves_c1 to c5, inferred via covariation modeling. e, Integration efficiency of R2Tgv1.1 with donor RNAs harboring modified 5’ UTR sequences in HEK293T cells. Sub1–10 denotes substitution of the first 1–10 nt of the 5’ UTR with heterologous sequences to disrupt the J1/2 secondary structure. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test. f, Length distribution of the buffer region in the avian 3’ UTR. g, Nucleotide composition of the buffer region in the avian 3’ UTR. For all graphs, data are represented as mean ± SD from three biological replicates.

    Extended Data Fig. 5 Systematic comparison of the R2 tool design strategy developed in this study with those of previously reported R2 tools.

    a, Summary of the designs of engineered R2 retrotransposons in this study and two prior studies (Study 17 and Study 26). Mini-donor from study2 was used for comparison. St1, study1; St2, study2. b, Comparison of gene integration activity of R2 variants from different designs, with cross-combination validation of R2 proteins and donor RNAs in HEK293T cells. mRNAs encoding R2Tg shared identical codon usage, translational UTRs and poly(A) tail lengths; donor RNAs were constructed as described in the original reports, including their native promoters, terminators and transgene expression cassette orientations. c, Schematic diagram of donor RNAs with varying lengths. d-e, Comparison of the gene integration capacity of R2 tools from different designs using RNA donors with elongated stuffer sequences (d) or RNA donors carrying gene-T2A-GFP expression cassettes (e) in HEK293T cells. f, Schematic of the RNA donors with dual-fluorescence reporter system (GFP and mCherry) for different R2 tools. g, Gating strategy for flow cytometric analysis of dual fluorescence. h, Assessment of the dual-fluorescence gene integration efficiency of R2 tools from different designs. i, Comparison of the integrity of gene integration products mediated by R2 tools from different designs. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test. For all graphs, data are represented as mean ± SD from three biological replicates.

    Extended Data Fig. 6 Investigation of the factors influencing differential activity among R2 tools designs.

    a, Replacement of the 5’ UTR in R2Tg donorvx with that derived from Tribolium castaneum significantly reduced the gene integration activity in HEK293T cells. Unpaired, two-tailed Student’s t test. b, Evaluation of integration efficiency mediated by R2Tgv1.1, where donorvx carried 28-nt, 36-nt, or 100-nt left homology arms (LHA) with or without the 5’ UTR and 5’ Cap-1 in different experimental groups in HEK293T cells. c, Analysis of integration efficiency mediated by R2Tgv1.1 with donors harboring different transgene expression constructs (all-RNA system) in HEK293T cells. Reverse constructs: expression cassette opposite to rDNA transcription direction; Forward constructs: cassette aligned with rDNA transcription direction. 1–9 represent different promoter-polyA terminator combinations. MFI: mean fluorescence intensity of GFP+ cells. d, ddPCR quantification of gene integration copy numbers corresponding to the distinct RNA donor constructs in Panel C. e, Correlation analysis between transgene copy number and fluorescence intensity for all constructs in Panel C via linear regression. Fluorescence intensity of each sample was calculated as MFI multiplied by the percentage of GFP+ cells, reflecting the relative total fluorescence intensity of each sample. f, Correlation analysis between transgene copy number and fluorescence intensity for CMV-containing constructs in Panel C via linear regression. g, Percentage and MFI of GFP+ cells after transfection of corresponding IVT-donor plasmids using Lipofectamine 3000 in HEK293T cells. For all graphs, data are represented as mean ± SD from three biological replicates.

    Extended Data Fig. 7 Systematic characterization of the R2 tools in terms of transgene copy number, integration specificity and full-length insertion capacity in HEK293T cells.

    a, R2 transgene integration copy numbers in the human genome, quantified via ddPCR. Unpaired, two-tailed Student’s t tests. b, Linear regression analysis of total fluorescence intensity versus corresponding transgene copy numbers. Fluorescence intensity of each sample was calculated as MFI multiplied by the percentage of GFP+ cells, reflecting the relative total fluorescence intensity of each sample. c, Whole-genome sequencing showing high on-target integration specificity of R2 retrotransposons in mammalian cells. d, Assessment of the integrity of R2-integrated transgene via whole-genome sequencing. e–j, Investigation of factors influencing the full-length transgene insertion proportion of the R2 retrotransposon tool using the GFP-T2A-mCherry dual-fluorescence reporter system; (e) Percentage of fluorescence-positive cells and (f) proportion of full-length transgene insertions (assessing effects of donor designs). FC, forward construct. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test. (g) Percentage of fluorescence-positive cells and (h) proportion of full-length transgene insertions (assessing effects of R2Tg protein designs). Unpaired, two-tailed Student’s t tests. (i) Percentage of fluorescence-positive cells and (j) proportion of full-length transgene insertions (assessing effects of delivery reagents). Unpaired, two-tailed Student’s t tests. Unless otherwise stated, data are presented as mean ± standard deviation (SD) from three biological replicates.

    Extended Data Fig. 8 Long-term monitoring of transgene expression mediated by R2 integration in HEK293T cells.

    a, Percentage (left) and MFI (right) of GFP+ cells during long-term continuous culture after R2-mediated integration. b, Copy numbers of integrated transgenes during long-term culture (from Extended Data Fig. 8a). Paired, two-tailed Student’s t test. c, Mixed culture of GFP+ cells (from Extended Data Fig. 8a) and negative cells (1:1 ratio) shows faster decline in GFP+ percentage than predicted. Predicted GFP+ percentage: half the positive value from Extended Data Fig. 8a at corresponding days; predicted MFI: MFI of GFP+ cells from Extended Data Fig. 8a. d, CRISPR-Cas9-induced rDNA recombination reduces the percentage and MFI of GFP+ cells; notably, nCas9(H840A)+sg1 did not induce a significant GFP+% and MFI reduction. e–f, Targeted deep sequencing analysis of indel frequencies at rDNA loci induced by the endonuclease activity of R2 retrotransposons (e) or Cas9 (f). Indel frequencies were defined as the proportion of rDNA loci containing indels relative to the total rDNA pool, normalized by subtracting background values from negative controls. g, Sorted GFP+ cells (from Extended Data Fig. 8a) sustain stable and high-efficiency transgene expression during long-term culture. h-i, Transfection of R2 protein-encoding mRNA into stably GFP-expressing cells (derived from Extended Data Fig. 8 g) induced indel formation ( < 0.1%) at rDNA loci (h); however, this did not compromise the percentage of GFP+ cells or MFI (i). END means R2 protein with inactivated active site of RLE domain; RTD means R2 protein with inactivated active site of the RT domain. j–k, Investigation of the effects of different mass ratios of R2 protein-encoding mRNA and donor RNA on transgene expression stability, with the total delivered mass maintained at 1.5 μg. Donor RNA carrying the CMV-GFP transgene (j) and CMV-scFv-T2A-GFP transgene (k) were tested. The R2Tgv1.1 R1119A mutant, derived from the previously described “endoculease-tuned” variants7, was included; residue 1119 corresponds to the amino acid numbering of wild-type R2Tg. All timelines described above are based on Extended Data Fig. 8a. Unless otherwise stated, data are presented as mean ± standard deviation (SD) from three (j–k) or five (a–i) biological replicates.

    Extended Data Fig. 9 Systematic investigation of gene integration mediated by newly mined and engineered R2SPs retrotransposons in primary T cells.

    a, Screening of viral proteins (Vp1 to Vp6) to enhance R2-mediated integration activity in T cells. Paired, two-tailed Student’s t test. b–g, Systematic assessment of the effects of the R2 tool and Vp4 (MERS-CoV-ORF4a) on primary T cell, with relevant parameters quantified at specified time points. (b) Percentage of GFP+ T cells with GFP gene integration at Day 2; (c) MFI of GFP+ T cells with GFP gene integration at Day 2; (d) Viable cell numbers over input at Day 2 post-electroporation; (e) Cell expansion fold-change at Day 10 relative to Day 2; (f) T cell viability rate at Day 2; (g) T cell viability rate at Day 10. END means R2 protein with inactivated active site of the endonuclease domain; RTD means R2 protein with inactivated active site of the RT domain; Q9P means Vp4 protein with inactivated active site of the RNA-binding domain23. h–i, Whole-genome sequencing demonstrating that Vp4 exerts minimal effects on R2 retrotransposons integration specificity (h) and full-length insertion (g) in human primary T cells. Data were obtained using Day 2 samples (from Extended Data Fig. 9b). j, ddPCR quantification of transgene copy numbers in T cells treated by different components (data collected at Day 2). k, long-term monitoring of transgene copy number changes in T cells via ddPCR. l, Integration of longer RNA donors (CMV-scFv-T2A-GFP) in primary T cells using the R2SPsv1.2 tool; long-term detection of GFP+ cell percentage and MFI. m, long-term monitoring of transgene copy number changes in T cells with integrated longer transgene (corresponding to Extended Data Fig. 9 l) via ddPCR. n, Assessment of DNA methylation dynamics at the CMV promoter of integrated transgenes in Day 2 and Day 12 samples (from Extended Data Fig. 9 l) via bisulfite sequencing. o, Investigation of the effects of transgene constructs with distinct promoters or transgene-expressing orientation on transgene expression stability in primary T cells. p, Palbociclib treatment attenuates the decline in GFP+% and GFP signal intensity. q, Palbociclib treatment reduces the proliferation rate of primary T cells. r–s, Integration of the CAR-CD19 transgene in primary T cells using the engineered R2SPsv1.2 tool. (r) Percentage of CAR-CD19-positive cells at Day 2; (s) Percentage of CAR-CD19-positive cells at Day 10. t, Cytotoxicity assay of R2-edited CAR-T cells via coculture with target tumor cells. Unless otherwise stated, data are presented as mean SD from 3 biological replicates from different T cell donors.

    Extended Data Fig. 10 Systematic investigation of gene integration mediated by newly mined and engineered R2SPs retrotransposons in human primary NK cells.

    a–f, Systematic assessment of the effects of the R2 tool and Vp4 on human primary NK cell status, with relevant parameters quantified at specified time points. (a) Dynamic changes in GFP+ cell percentage and MFI of NK cells with GFP gene integration from Day 2 to Day 6 (a subset of R2-treated NK cells was reactivated at Day 2); (b) Monitoring transgene copy numbers in NK cells via ddPCR; (c) Viable cell numbers at Day 2 post-electroporation; (d) Cell expansion fold-change at Day 6 relative to Day 2; (e) NK cell viability rate at Day 2; (f) NK cell viability rate at Day 6; n = 3 biological replicates from different NK cell donors. g, Integration of RNA donor harboring CMV-GFP transgene in primary human foreskin fibroblasts using the R2SPsv1.2 tool; long-term detection of GFP+ cell percentage and MFI. n = 3 biological replicates. h, Monitoring transgene copy numbers in HFFs via ddPCR. Unless otherwise stated, data are presented as mean ± SD.

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    Chen, Y., Hu, Y., Luo, S. et al. Discovery and engineering of avian R2 retrotransposons for all-RNA-mediated targeted DNA integration in human cells. Nat Biotechnol (2026). https://doi.org/10.1038/s41587-026-03315-w

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