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Data schedule 19460578 The sequencing information associated with this research study have actually been sent to the Genome Sequence Archive for Human, a platform under the Beijing Institute of Genomics, Chinese Academy of Sciences (offered at https://ngdc.cncb.ac.cn/gsa-human/ 19460581. Since the paper’s publication date, the information will be open to the general public, and the accession number for retrieval is HRA013312 19460581 (ref. 48 19460584. Source information are supplied with this paper. 19460585
Code Availability 19460578 19460579 Analytic code for the sequencing information is openly offered at GitHub at https://github.com/YanpingHu/avian_R2 (ref. 49. 19460586
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GitHub https://github.com/YanpingHu/avian_R2 19460581 (2026). 19460585 19460877 19460579 Download referrals 19460585
Acknowledgements 19460578 19460579 We want to thank J. Sun for assistance on T cell control. We likewise thank the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, for offering the main HHFs utilized in this research study. 19460586
Funding 19460578 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. 19460585 19460586
Author info 19460578 Author notes 19460887 19460888 19460579 These authors contributed similarly: Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang.
- 19460585 19460602 Authors and Affiliations 19460888 19460579 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 19460579 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 19460585 19460602 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China 19460585 Yangcan Chen, Yanping Hu, Na Tang & , Chenxin Wang, Xinge Wang, Peng Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li 19460585 19460602 19460579 Authors 19460585 Yangcan Chen 19460887 19460896 Yanping Hu 19460887 Shengqiu Luo 19460887 19460896 Bangwei Mao 19460602 Na Tang 19460602 Chenxin Wang 19460887 19460896 Yuze Wang 19460887 Hanyu Bai Xinge Wang 19460887 19460896 Changxian Peng 19460887 19460602 Qicheng Chen 19460887 19460602 19460896 Jiahui Xiao 19460602 Yu Zou 19460602 19460896 Peng Wang Rui Niu 19460602 19460896 Jin Zhang 19460602 19460896 Yulong Zhao Chen Liang Yunlong Wang 19460887 Yiyun Li 19460887 19460896 Junjie Wang 19460887 Ng Shyh-Chang 19460602 19460896 Tuo Wei 19460887 19460602 Haoyi Wang 19460887 19460602 19460896 Qi Zhou 19460896 Wei Li 19460602 19460586 Contributions W.L., Q.Z. and Y.C. developed the task and created 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. carried out the experiments. W.L., Q.Z., Y.C., Y.H. and S.L. evaluated the information. W.L., Q.Z., Y.C. and H.W. monitored the research study. W.L., Q.Z., Y.C., Y.H., S.L., N.S.-C., T.W. and H.W. composed the paper, helped by the other authors. 19460585 Corresponding authors 19460891 Correspondence to Yangcan Chen, Haoyi Wang, Qi Zhou or Wei Li. 19460586
Ethics statements 19460578 Contending interests 19460891 W.L., Q.Z., Y.C., Y.H., S.L., Q.C., X.W. and B.M. have actually sent patent applications associated to this paper. The other authors state no contending interests. 19460586 19460586
Peer evaluation Peer evaluation info 19460579 19460597 Nature Biotechnology 19460598 thanks Lei Qi, Max Wilkinson and the other, confidential, customer(s )for their contribution to the peer evaluation of this work.
19460586 Additional info 19460578 Publisher’s note Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19460585
Extended information 19460940 Extended Data Fig. 1 Workflow for mining brand-new R2 retrotransposons in bird genomes. 19460891 19460599 a , Computational pipeline for determining bird R2 retrotransposons. 19460599 b , Distribution of freshly found R2 aspects throughout orders of evaluated bird genomes. 19460599 c , Left: circulation of downloaded bird genomes throughout various orders; Right: circulation of bird genomes consisting of full-length R2 components throughout various orders. 19460585 Extended Data Fig. 2 Systematic characterization of Region-1 and Region-2 in the R2Tg protein. 19460579 a 19460600, Droplet digital PCR (ddPCR)analysis shows that Region-1 truncation considerably hinders the molecular combination effectiveness of R2Tg, and this flaw can be saved by HMGN1 blend. Cp, copies. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. b 19460600, Effect of Region-1 truncation length on R2-mediated gene combination performance in HEK293T cells, paired with donor 19460583 choose 19460584 c 19460600, Identification of replacing domains capable for saving Region-1 function in HEK293T cells, combined with donor choose NLS1/2, Nuclear Localization Signal 1/2 17 ; NBD, nucleosome-binding domain; CHUD, chromatin unfolding domain. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 d , Trans-expression of Region-1 stops working to save R2-mediated gene combination effectiveness in HEK293T cells, combined with donor choose 19460584 Unpaired, two-tailed Student’s t tests. e 19460600, Impact of region-2 truncation on R2-mediated gene combination activity in HEK293T cells, combined with donor decide 19460584 Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 f , Statistical analysis of Region-1 length throughout 165 bird R2 retrotransposon proteins. 19460599 g , Statistical analysis of length of unique subregions (Spacer, NTE-2 and partial NTE-1) in Region-2 throughout 165 bird R2 retrotransposon proteins. h 19460600, Evaluating the activity of R2Tg protein with gotten rid of nuclear localization signal or rearranged Flag tag in all-RNA speculative systems in HEK293T cells, paired with donor decide 19460584 Unless otherwise mentioned, information exist as mean ± basic variance (SD) from 3 biological reproduces. 19460585 Extended Data Fig. 3 Investigation of the impacts of donor RHA length and polyA length on R2-mediated gene combination effectiveness. 19460891 19460579 a , Impact of donor RNA with differing best homology arm (RHA)and polyA lengths on combination performance in HEK293T cells. United States, unassociated vector series. 19460599 b 19460600, Impact of donor RNA with 4 nt RHA and different polyA lengths in HEK293T cells. c 19460600, Integration performance of R2Tg v1.1 19460584 with donor RNAs including various nucleotide elements after a 4 bp RHA in HEK293T cells. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates. 19460585 19460940 Extended Data Fig. 4 Investigation of the results of 5’UTR components on R2-mediated gene combination performance. 19460891 a , Statistical analysis of J1/2 area length throughout 165 bird R2 5’ribozymes. 19460599 b 19460600, Predicted secondary structure of the J1/2 area in 165 bird R2 5 ‘ribozymes, presumed through covariation modeling. 19460599 c 19460600, Statistical analysis of the length of non-ribozyme part in 165 R2 5’UTRs. d , Predicted secondary structure of the non-ribozyme part in bird R2 5’UTRs from Aves_c1 to c5, presumed through covariation modeling. 19460599 e , Integration performance of R2Tg 19460583 v1.1 with donor RNAs harboring customized 5′ UTR series in HEK293T cells. Sub1– 10 represents replacement of the very first 1– 10 nt of the 5′ UTR with heterologous series to interrupt the J1/2 secondary structure. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. 19460599 f , Length circulation of the buffer area in the bird 3′ UTR. g 19460600, Nucleotide structure of the buffer area in the bird 3’ UTR. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460586 19460940 Extended Data Fig. 5 Systematic contrast of the R2 tool style method established in this research study with those of formerly reported R2 tools. 19460891 19460599 a 19460600, Summary of the styles of crafted R2 retrotransposons in this research study and 2 previous research studies (Study 1 19460583 7 and Study 2 6. Mini-donor from study2 was utilized for contrast. St1, study1; St2, study2. 19460599 b , Comparison of gene combination activity of R2 versions from various styles, with cross-combination recognition of R2 proteins and donor RNAs in HEK293T cells. mRNAs encoding R2Tg shared similar codon use, translational UTRs and poly(A) tail lengths; donor RNAs were built as explained in the initial reports, including their native promoters, terminators and transgene expression cassette orientations. c 19460600, Schematic diagram of donor RNAs with differing lengths. d-e 19460600, Comparison of the gene combination capability of R2 tools from various styles utilizing RNA donors with lengthened stuffer series ( 19460599 d 19460600 or RNA donors bring gene-T2A-GFP expression cassettes ( 19460599 e 19460600 in HEK293T cells. f 19460600, Schematic of the RNA donors with dual-fluorescence press reporter system (GFP and mCherry) for various R2 tools. g 19460600, Gating technique for circulation cytometric analysis of double fluorescence. 19460599 h , Assessment of the dual-fluorescence gene combination effectiveness of R2 tools from various styles. 19460599 i 19460600, Comparison of the stability of gene combination items moderated by R2 tools from various styles. Analytical analyses were carried out utilizing one-way ANOVA with Tukey’s several contrasts test. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460585
19460940 Extended Data Fig. 6 Investigation of the elements affecting differential activity amongst R2 tools styles. a , Replacement of the 5′ UTR in R2Tg donor vx 19460584 with that originated from 19460597 Tribolium castaneum 19460598 considerably decreased the gene combination activity in HEK293T cells. Unpaired, two-tailed Student’s t test. 19460599 b 19460600, Evaluation of combination effectiveness moderated by R2Tg v1.1 , where donor 19460583 vx brought 28-nt, 36-nt, or 100-nt left homology arms (LHA) with or without the 5′ UTR and 5′ Cap-1 in various speculative groups in HEK293T cells. c , Analysis of combination performance moderated by R2Tg v1.1 with donors harboring various transgene expression constructs (all-RNA system) in HEK293T cells. Reverse constructs: expression cassette opposite to rDNA transcription instructions; Forward constructs: cassette lined up with rDNA transcription instructions. 1– 9 represent various promoter-polyA terminator mixes. MFI: indicate fluorescence strength of GFP + cells. 19460599 d , ddPCR metrology of gene combination copy numbers representing the unique RNA donor constructs in Panel C.eCorrelation analysis in between transgene copy number and fluorescence strength for all constructs in Panel C by means of direct regression. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP +cells, showing the relative overall fluorescence strength of each sample.fCorrelation analysis in between transgene copy number and fluorescence strength for CMV-containing constructs in Panel C through direct regression.gPercentage and MFI of GFP +cells after transfection of matching IVT-donor plasmids utilizing Lipofectamine 3000 in HEK293T cells. For all charts, information are represented as mean ± SD from 3 biological reproduces.
Extended Data Fig. 7 Systematic characterization of the R2 tools in regards to transgene copy number, combination uniqueness and full-length insertion capability in HEK293T cells.
aR2 transgene combination copy numbers in the human genome, measured by means of ddPCR. Unpaired, two-tailed Student’s t tests.bLinear regression analysis of overall fluorescence strength versus matching transgene copy numbers. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP+cells, showing the relative overall fluorescence strength of each sample.cWhole-genome sequencing revealing high on-target combination uniqueness of R2 retrotransposons in mammalian cells.dAssessment of the stability of R2-integrated transgene by means of whole-genome sequencing.e– jInvestigation of elements affecting the full-length transgene insertion percentage of the R2 retrotransposon tool utilizing the GFP-T2A-mCherry dual-fluorescence press reporter system;(e)Portion of fluorescence-positive cells and(f)percentage of full-length transgene insertions (examining results of donor styles). FC, forward construct. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test.(g)Portion of fluorescence-positive cells and(h)percentage of full-length transgene insertions (evaluating results of R2Tg protein styles). Unpaired, two-tailed Student’s t tests.(i)Portion of fluorescence-positive cells and(j)percentage of full-length transgene insertions (evaluating results of shipment reagents). Unpaired, two-tailed Student’s t tests. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates.
Extended Data Fig. 8 Long-term tracking of transgene expression moderated by R2 combination in HEK293T cells.
aPercentage(left)and MFI(ideal)of GFP+cells throughout long-lasting constant culture after R2-mediated combination.bCopy varieties of incorporated transgenes throughout long-lasting culture( from Extended Data Fig. 8a). Paired, two-tailed Student’s t test.cMixed culture of GFP+cells(from Extended Data Fig. 8a )and unfavorable cells(1:1 ratio) reveals faster decrease in GFP+portion than forecasted. Anticipated GFP+portion: half the favorable worth from Extended Data Fig. 8a at matching days; anticipated MFI: MFI of GFP+cells from Extended Data Fig. 8a.dCRISPR-Cas9-induced rDNA recombination minimizes the portion and MFI of GFP+cells; especially, nCas9(H840A)+sg1 did not cause a substantial GFP+% and MFI decrease.e– fTargeted deep sequencing analysis of indel frequencies at rDNA loci caused by the endonuclease activity of R2 retrotransposons (eor Cas9 (f. Indel frequencies were specified as the percentage of rDNA loci including indels relative to the overall rDNA swimming pool, stabilized by deducting background worths from unfavorable controls.gSorted GFP+ cells (from Extended Data Fig. 8a) sustain steady and high-efficiency transgene expression throughout long-lasting culture.h-iTransfection of R2 protein-encoding mRNA into stably GFP-expressing cells (originated from Extended Data Fig. 8 g) caused indel development (
Code Availability 19460578 19460579 Analytic code for the sequencing information is openly offered at GitHub at https://github.com/YanpingHu/avian_R2 (ref. 49. 19460586
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GitHub https://github.com/YanpingHu/avian_R2 19460581 (2026). 19460585 19460877 19460579 Download referrals 19460585
Acknowledgements 19460578 19460579 We want to thank J. Sun for assistance on T cell control. We likewise thank the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, for offering the main HHFs utilized in this research study. 19460586
Funding 19460578 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. 19460585 19460586
Author info 19460578 Author notes 19460887 19460888 19460579 These authors contributed similarly: Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang.
- 19460585 19460602 Authors and Affiliations 19460888 19460579 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 19460579 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 19460585 19460602 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China 19460585 Yangcan Chen, Yanping Hu, Na Tang & , Chenxin Wang, Xinge Wang, Peng Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li 19460585 19460602 19460579 Authors 19460585 Yangcan Chen 19460887 19460896 Yanping Hu 19460887 Shengqiu Luo 19460887 19460896 Bangwei Mao 19460602 Na Tang 19460602 Chenxin Wang 19460887 19460896 Yuze Wang 19460887 Hanyu Bai Xinge Wang 19460887 19460896 Changxian Peng 19460887 19460602 Qicheng Chen 19460887 19460602 19460896 Jiahui Xiao 19460602 Yu Zou 19460602 19460896 Peng Wang Rui Niu 19460602 19460896 Jin Zhang 19460602 19460896 Yulong Zhao Chen Liang Yunlong Wang 19460887 Yiyun Li 19460887 19460896 Junjie Wang 19460887 Ng Shyh-Chang 19460602 19460896 Tuo Wei 19460887 19460602 Haoyi Wang 19460887 19460602 19460896 Qi Zhou 19460896 Wei Li 19460602 19460586 Contributions W.L., Q.Z. and Y.C. developed the task and created 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. carried out the experiments. W.L., Q.Z., Y.C., Y.H. and S.L. evaluated the information. W.L., Q.Z., Y.C. and H.W. monitored the research study. W.L., Q.Z., Y.C., Y.H., S.L., N.S.-C., T.W. and H.W. composed the paper, helped by the other authors. 19460585 Corresponding authors 19460891 Correspondence to Yangcan Chen, Haoyi Wang, Qi Zhou or Wei Li. 19460586
Ethics statements 19460578 Contending interests 19460891 W.L., Q.Z., Y.C., Y.H., S.L., Q.C., X.W. and B.M. have actually sent patent applications associated to this paper. The other authors state no contending interests. 19460586 19460586
Peer evaluation Peer evaluation info 19460579 19460597 Nature Biotechnology 19460598 thanks Lei Qi, Max Wilkinson and the other, confidential, customer(s )for their contribution to the peer evaluation of this work.
19460586 Additional info 19460578 Publisher’s note Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19460585
Extended information 19460940 Extended Data Fig. 1 Workflow for mining brand-new R2 retrotransposons in bird genomes. 19460891 19460599 a , Computational pipeline for determining bird R2 retrotransposons. 19460599 b , Distribution of freshly found R2 aspects throughout orders of evaluated bird genomes. 19460599 c , Left: circulation of downloaded bird genomes throughout various orders; Right: circulation of bird genomes consisting of full-length R2 components throughout various orders. 19460585 Extended Data Fig. 2 Systematic characterization of Region-1 and Region-2 in the R2Tg protein. 19460579 a 19460600, Droplet digital PCR (ddPCR)analysis shows that Region-1 truncation considerably hinders the molecular combination effectiveness of R2Tg, and this flaw can be saved by HMGN1 blend. Cp, copies. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. b 19460600, Effect of Region-1 truncation length on R2-mediated gene combination performance in HEK293T cells, paired with donor 19460583 choose 19460584 c 19460600, Identification of replacing domains capable for saving Region-1 function in HEK293T cells, combined with donor choose NLS1/2, Nuclear Localization Signal 1/2 17 ; NBD, nucleosome-binding domain; CHUD, chromatin unfolding domain. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 d , Trans-expression of Region-1 stops working to save R2-mediated gene combination effectiveness in HEK293T cells, combined with donor choose 19460584 Unpaired, two-tailed Student’s t tests. e 19460600, Impact of region-2 truncation on R2-mediated gene combination activity in HEK293T cells, combined with donor decide 19460584 Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 f , Statistical analysis of Region-1 length throughout 165 bird R2 retrotransposon proteins. 19460599 g , Statistical analysis of length of unique subregions (Spacer, NTE-2 and partial NTE-1) in Region-2 throughout 165 bird R2 retrotransposon proteins. h 19460600, Evaluating the activity of R2Tg protein with gotten rid of nuclear localization signal or rearranged Flag tag in all-RNA speculative systems in HEK293T cells, paired with donor decide 19460584 Unless otherwise mentioned, information exist as mean ± basic variance (SD) from 3 biological reproduces. 19460585 Extended Data Fig. 3 Investigation of the impacts of donor RHA length and polyA length on R2-mediated gene combination effectiveness. 19460891 19460579 a , Impact of donor RNA with differing best homology arm (RHA)and polyA lengths on combination performance in HEK293T cells. United States, unassociated vector series. 19460599 b 19460600, Impact of donor RNA with 4 nt RHA and different polyA lengths in HEK293T cells. c 19460600, Integration performance of R2Tg v1.1 19460584 with donor RNAs including various nucleotide elements after a 4 bp RHA in HEK293T cells. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates. 19460585 19460940 Extended Data Fig. 4 Investigation of the results of 5’UTR components on R2-mediated gene combination performance. 19460891 a , Statistical analysis of J1/2 area length throughout 165 bird R2 5’ribozymes. 19460599 b 19460600, Predicted secondary structure of the J1/2 area in 165 bird R2 5 ‘ribozymes, presumed through covariation modeling. 19460599 c 19460600, Statistical analysis of the length of non-ribozyme part in 165 R2 5’UTRs. d , Predicted secondary structure of the non-ribozyme part in bird R2 5’UTRs from Aves_c1 to c5, presumed through covariation modeling. 19460599 e , Integration performance of R2Tg 19460583 v1.1 with donor RNAs harboring customized 5′ UTR series in HEK293T cells. Sub1– 10 represents replacement of the very first 1– 10 nt of the 5′ UTR with heterologous series to interrupt the J1/2 secondary structure. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. 19460599 f , Length circulation of the buffer area in the bird 3′ UTR. g 19460600, Nucleotide structure of the buffer area in the bird 3’ UTR. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460586 19460940 Extended Data Fig. 5 Systematic contrast of the R2 tool style method established in this research study with those of formerly reported R2 tools. 19460891 19460599 a 19460600, Summary of the styles of crafted R2 retrotransposons in this research study and 2 previous research studies (Study 1 19460583 7 and Study 2 6. Mini-donor from study2 was utilized for contrast. St1, study1; St2, study2. 19460599 b , Comparison of gene combination activity of R2 versions from various styles, with cross-combination recognition of R2 proteins and donor RNAs in HEK293T cells. mRNAs encoding R2Tg shared similar codon use, translational UTRs and poly(A) tail lengths; donor RNAs were built as explained in the initial reports, including their native promoters, terminators and transgene expression cassette orientations. c 19460600, Schematic diagram of donor RNAs with differing lengths. d-e 19460600, Comparison of the gene combination capability of R2 tools from various styles utilizing RNA donors with lengthened stuffer series ( 19460599 d 19460600 or RNA donors bring gene-T2A-GFP expression cassettes ( 19460599 e 19460600 in HEK293T cells. f 19460600, Schematic of the RNA donors with dual-fluorescence press reporter system (GFP and mCherry) for various R2 tools. g 19460600, Gating technique for circulation cytometric analysis of double fluorescence. 19460599 h , Assessment of the dual-fluorescence gene combination effectiveness of R2 tools from various styles. 19460599 i 19460600, Comparison of the stability of gene combination items moderated by R2 tools from various styles. Analytical analyses were carried out utilizing one-way ANOVA with Tukey’s several contrasts test. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460585
19460940 Extended Data Fig. 6 Investigation of the elements affecting differential activity amongst R2 tools styles. a , Replacement of the 5′ UTR in R2Tg donor vx 19460584 with that originated from 19460597 Tribolium castaneum 19460598 considerably decreased the gene combination activity in HEK293T cells. Unpaired, two-tailed Student’s t test. 19460599 b 19460600, Evaluation of combination effectiveness moderated by R2Tg v1.1 , where donor 19460583 vx brought 28-nt, 36-nt, or 100-nt left homology arms (LHA) with or without the 5′ UTR and 5′ Cap-1 in various speculative groups in HEK293T cells. c , Analysis of combination performance moderated by R2Tg v1.1 with donors harboring various transgene expression constructs (all-RNA system) in HEK293T cells. Reverse constructs: expression cassette opposite to rDNA transcription instructions; Forward constructs: cassette lined up with rDNA transcription instructions. 1– 9 represent various promoter-polyA terminator mixes. MFI: indicate fluorescence strength of GFP + cells. 19460599 d , ddPCR metrology of gene combination copy numbers representing the unique RNA donor constructs in Panel C.eCorrelation analysis in between transgene copy number and fluorescence strength for all constructs in Panel C by means of direct regression. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP +cells, showing the relative overall fluorescence strength of each sample.fCorrelation analysis in between transgene copy number and fluorescence strength for CMV-containing constructs in Panel C through direct regression.gPercentage and MFI of GFP +cells after transfection of matching IVT-donor plasmids utilizing Lipofectamine 3000 in HEK293T cells. For all charts, information are represented as mean ± SD from 3 biological reproduces.
Extended Data Fig. 7 Systematic characterization of the R2 tools in regards to transgene copy number, combination uniqueness and full-length insertion capability in HEK293T cells.
aR2 transgene combination copy numbers in the human genome, measured by means of ddPCR. Unpaired, two-tailed Student’s t tests.bLinear regression analysis of overall fluorescence strength versus matching transgene copy numbers. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP+cells, showing the relative overall fluorescence strength of each sample.cWhole-genome sequencing revealing high on-target combination uniqueness of R2 retrotransposons in mammalian cells.dAssessment of the stability of R2-integrated transgene by means of whole-genome sequencing.e– jInvestigation of elements affecting the full-length transgene insertion percentage of the R2 retrotransposon tool utilizing the GFP-T2A-mCherry dual-fluorescence press reporter system;(e)Portion of fluorescence-positive cells and(f)percentage of full-length transgene insertions (examining results of donor styles). FC, forward construct. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test.(g)Portion of fluorescence-positive cells and(h)percentage of full-length transgene insertions (evaluating results of R2Tg protein styles). Unpaired, two-tailed Student’s t tests.(i)Portion of fluorescence-positive cells and(j)percentage of full-length transgene insertions (evaluating results of shipment reagents). Unpaired, two-tailed Student’s t tests. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates.
Extended Data Fig. 8 Long-term tracking of transgene expression moderated by R2 combination in HEK293T cells.
aPercentage(left)and MFI(ideal)of GFP+cells throughout long-lasting constant culture after R2-mediated combination.bCopy varieties of incorporated transgenes throughout long-lasting culture( from Extended Data Fig. 8a). Paired, two-tailed Student’s t test.cMixed culture of GFP+cells(from Extended Data Fig. 8a )and unfavorable cells(1:1 ratio) reveals faster decrease in GFP+portion than forecasted. Anticipated GFP+portion: half the favorable worth from Extended Data Fig. 8a at matching days; anticipated MFI: MFI of GFP+cells from Extended Data Fig. 8a.dCRISPR-Cas9-induced rDNA recombination minimizes the portion and MFI of GFP+cells; especially, nCas9(H840A)+sg1 did not cause a substantial GFP+% and MFI decrease.e– fTargeted deep sequencing analysis of indel frequencies at rDNA loci caused by the endonuclease activity of R2 retrotransposons (eor Cas9 (f. Indel frequencies were specified as the percentage of rDNA loci including indels relative to the overall rDNA swimming pool, stabilized by deducting background worths from unfavorable controls.gSorted GFP+ cells (from Extended Data Fig. 8a) sustain steady and high-efficiency transgene expression throughout long-lasting culture.h-iTransfection of R2 protein-encoding mRNA into stably GFP-expressing cells (originated from Extended Data Fig. 8 g) caused indel development (
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GitHub https://github.com/YanpingHu/avian_R2 19460581 (2026). 19460585 19460877 19460579 Download referrals 19460585
Acknowledgements 19460578 19460579 We want to thank J. Sun for assistance on T cell control. We likewise thank the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, for offering the main HHFs utilized in this research study. 19460586
Funding 19460578 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. 19460585 19460586
Author info 19460578 Author notes 19460887 19460888 19460579 These authors contributed similarly: Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang.
- 19460585 19460602 Authors and Affiliations 19460888 19460579 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 19460579 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 19460585 19460602 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China 19460585 Yangcan Chen, Yanping Hu, Na Tang & , Chenxin Wang, Xinge Wang, Peng Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li 19460585 19460602 19460579 Authors 19460585 Yangcan Chen 19460887 19460896 Yanping Hu 19460887 Shengqiu Luo 19460887 19460896 Bangwei Mao 19460602 Na Tang 19460602 Chenxin Wang 19460887 19460896 Yuze Wang 19460887 Hanyu Bai Xinge Wang 19460887 19460896 Changxian Peng 19460887 19460602 Qicheng Chen 19460887 19460602 19460896 Jiahui Xiao 19460602 Yu Zou 19460602 19460896 Peng Wang Rui Niu 19460602 19460896 Jin Zhang 19460602 19460896 Yulong Zhao Chen Liang Yunlong Wang 19460887 Yiyun Li 19460887 19460896 Junjie Wang 19460887 Ng Shyh-Chang 19460602 19460896 Tuo Wei 19460887 19460602 Haoyi Wang 19460887 19460602 19460896 Qi Zhou 19460896 Wei Li 19460602 19460586 Contributions W.L., Q.Z. and Y.C. developed the task and created 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. carried out the experiments. W.L., Q.Z., Y.C., Y.H. and S.L. evaluated the information. W.L., Q.Z., Y.C. and H.W. monitored the research study. W.L., Q.Z., Y.C., Y.H., S.L., N.S.-C., T.W. and H.W. composed the paper, helped by the other authors. 19460585 Corresponding authors 19460891 Correspondence to Yangcan Chen, Haoyi Wang, Qi Zhou or Wei Li. 19460586
Ethics statements 19460578 Contending interests 19460891 W.L., Q.Z., Y.C., Y.H., S.L., Q.C., X.W. and B.M. have actually sent patent applications associated to this paper. The other authors state no contending interests. 19460586 19460586
Peer evaluation Peer evaluation info 19460579 19460597 Nature Biotechnology 19460598 thanks Lei Qi, Max Wilkinson and the other, confidential, customer(s )for their contribution to the peer evaluation of this work.
19460586 Additional info 19460578 Publisher’s note Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19460585
Extended information 19460940 Extended Data Fig. 1 Workflow for mining brand-new R2 retrotransposons in bird genomes. 19460891 19460599 a , Computational pipeline for determining bird R2 retrotransposons. 19460599 b , Distribution of freshly found R2 aspects throughout orders of evaluated bird genomes. 19460599 c , Left: circulation of downloaded bird genomes throughout various orders; Right: circulation of bird genomes consisting of full-length R2 components throughout various orders. 19460585 Extended Data Fig. 2 Systematic characterization of Region-1 and Region-2 in the R2Tg protein. 19460579 a 19460600, Droplet digital PCR (ddPCR)analysis shows that Region-1 truncation considerably hinders the molecular combination effectiveness of R2Tg, and this flaw can be saved by HMGN1 blend. Cp, copies. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. b 19460600, Effect of Region-1 truncation length on R2-mediated gene combination performance in HEK293T cells, paired with donor 19460583 choose 19460584 c 19460600, Identification of replacing domains capable for saving Region-1 function in HEK293T cells, combined with donor choose NLS1/2, Nuclear Localization Signal 1/2 17 ; NBD, nucleosome-binding domain; CHUD, chromatin unfolding domain. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 d , Trans-expression of Region-1 stops working to save R2-mediated gene combination effectiveness in HEK293T cells, combined with donor choose 19460584 Unpaired, two-tailed Student’s t tests. e 19460600, Impact of region-2 truncation on R2-mediated gene combination activity in HEK293T cells, combined with donor decide 19460584 Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 f , Statistical analysis of Region-1 length throughout 165 bird R2 retrotransposon proteins. 19460599 g , Statistical analysis of length of unique subregions (Spacer, NTE-2 and partial NTE-1) in Region-2 throughout 165 bird R2 retrotransposon proteins. h 19460600, Evaluating the activity of R2Tg protein with gotten rid of nuclear localization signal or rearranged Flag tag in all-RNA speculative systems in HEK293T cells, paired with donor decide 19460584 Unless otherwise mentioned, information exist as mean ± basic variance (SD) from 3 biological reproduces. 19460585 Extended Data Fig. 3 Investigation of the impacts of donor RHA length and polyA length on R2-mediated gene combination effectiveness. 19460891 19460579 a , Impact of donor RNA with differing best homology arm (RHA)and polyA lengths on combination performance in HEK293T cells. United States, unassociated vector series. 19460599 b 19460600, Impact of donor RNA with 4 nt RHA and different polyA lengths in HEK293T cells. c 19460600, Integration performance of R2Tg v1.1 19460584 with donor RNAs including various nucleotide elements after a 4 bp RHA in HEK293T cells. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates. 19460585 19460940 Extended Data Fig. 4 Investigation of the results of 5’UTR components on R2-mediated gene combination performance. 19460891 a , Statistical analysis of J1/2 area length throughout 165 bird R2 5’ribozymes. 19460599 b 19460600, Predicted secondary structure of the J1/2 area in 165 bird R2 5 ‘ribozymes, presumed through covariation modeling. 19460599 c 19460600, Statistical analysis of the length of non-ribozyme part in 165 R2 5’UTRs. d , Predicted secondary structure of the non-ribozyme part in bird R2 5’UTRs from Aves_c1 to c5, presumed through covariation modeling. 19460599 e , Integration performance of R2Tg 19460583 v1.1 with donor RNAs harboring customized 5′ UTR series in HEK293T cells. Sub1– 10 represents replacement of the very first 1– 10 nt of the 5′ UTR with heterologous series to interrupt the J1/2 secondary structure. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. 19460599 f , Length circulation of the buffer area in the bird 3′ UTR. g 19460600, Nucleotide structure of the buffer area in the bird 3’ UTR. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460586 19460940 Extended Data Fig. 5 Systematic contrast of the R2 tool style method established in this research study with those of formerly reported R2 tools. 19460891 19460599 a 19460600, Summary of the styles of crafted R2 retrotransposons in this research study and 2 previous research studies (Study 1 19460583 7 and Study 2 6. Mini-donor from study2 was utilized for contrast. St1, study1; St2, study2. 19460599 b , Comparison of gene combination activity of R2 versions from various styles, with cross-combination recognition of R2 proteins and donor RNAs in HEK293T cells. mRNAs encoding R2Tg shared similar codon use, translational UTRs and poly(A) tail lengths; donor RNAs were built as explained in the initial reports, including their native promoters, terminators and transgene expression cassette orientations. c 19460600, Schematic diagram of donor RNAs with differing lengths. d-e 19460600, Comparison of the gene combination capability of R2 tools from various styles utilizing RNA donors with lengthened stuffer series ( 19460599 d 19460600 or RNA donors bring gene-T2A-GFP expression cassettes ( 19460599 e 19460600 in HEK293T cells. f 19460600, Schematic of the RNA donors with dual-fluorescence press reporter system (GFP and mCherry) for various R2 tools. g 19460600, Gating technique for circulation cytometric analysis of double fluorescence. 19460599 h , Assessment of the dual-fluorescence gene combination effectiveness of R2 tools from various styles. 19460599 i 19460600, Comparison of the stability of gene combination items moderated by R2 tools from various styles. Analytical analyses were carried out utilizing one-way ANOVA with Tukey’s several contrasts test. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460585
19460940 Extended Data Fig. 6 Investigation of the elements affecting differential activity amongst R2 tools styles. a , Replacement of the 5′ UTR in R2Tg donor vx 19460584 with that originated from 19460597 Tribolium castaneum 19460598 considerably decreased the gene combination activity in HEK293T cells. Unpaired, two-tailed Student’s t test. 19460599 b 19460600, Evaluation of combination effectiveness moderated by R2Tg v1.1 , where donor 19460583 vx brought 28-nt, 36-nt, or 100-nt left homology arms (LHA) with or without the 5′ UTR and 5′ Cap-1 in various speculative groups in HEK293T cells. c , Analysis of combination performance moderated by R2Tg v1.1 with donors harboring various transgene expression constructs (all-RNA system) in HEK293T cells. Reverse constructs: expression cassette opposite to rDNA transcription instructions; Forward constructs: cassette lined up with rDNA transcription instructions. 1– 9 represent various promoter-polyA terminator mixes. MFI: indicate fluorescence strength of GFP + cells. 19460599 d , ddPCR metrology of gene combination copy numbers representing the unique RNA donor constructs in Panel C.eCorrelation analysis in between transgene copy number and fluorescence strength for all constructs in Panel C by means of direct regression. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP +cells, showing the relative overall fluorescence strength of each sample.fCorrelation analysis in between transgene copy number and fluorescence strength for CMV-containing constructs in Panel C through direct regression.gPercentage and MFI of GFP +cells after transfection of matching IVT-donor plasmids utilizing Lipofectamine 3000 in HEK293T cells. For all charts, information are represented as mean ± SD from 3 biological reproduces.
Extended Data Fig. 7 Systematic characterization of the R2 tools in regards to transgene copy number, combination uniqueness and full-length insertion capability in HEK293T cells.
aR2 transgene combination copy numbers in the human genome, measured by means of ddPCR. Unpaired, two-tailed Student’s t tests.bLinear regression analysis of overall fluorescence strength versus matching transgene copy numbers. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP+cells, showing the relative overall fluorescence strength of each sample.cWhole-genome sequencing revealing high on-target combination uniqueness of R2 retrotransposons in mammalian cells.dAssessment of the stability of R2-integrated transgene by means of whole-genome sequencing.e– jInvestigation of elements affecting the full-length transgene insertion percentage of the R2 retrotransposon tool utilizing the GFP-T2A-mCherry dual-fluorescence press reporter system;(e)Portion of fluorescence-positive cells and(f)percentage of full-length transgene insertions (examining results of donor styles). FC, forward construct. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test.(g)Portion of fluorescence-positive cells and(h)percentage of full-length transgene insertions (evaluating results of R2Tg protein styles). Unpaired, two-tailed Student’s t tests.(i)Portion of fluorescence-positive cells and(j)percentage of full-length transgene insertions (evaluating results of shipment reagents). Unpaired, two-tailed Student’s t tests. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates.
Extended Data Fig. 8 Long-term tracking of transgene expression moderated by R2 combination in HEK293T cells.
aPercentage(left)and MFI(ideal)of GFP+cells throughout long-lasting constant culture after R2-mediated combination.bCopy varieties of incorporated transgenes throughout long-lasting culture( from Extended Data Fig. 8a). Paired, two-tailed Student’s t test.cMixed culture of GFP+cells(from Extended Data Fig. 8a )and unfavorable cells(1:1 ratio) reveals faster decrease in GFP+portion than forecasted. Anticipated GFP+portion: half the favorable worth from Extended Data Fig. 8a at matching days; anticipated MFI: MFI of GFP+cells from Extended Data Fig. 8a.dCRISPR-Cas9-induced rDNA recombination minimizes the portion and MFI of GFP+cells; especially, nCas9(H840A)+sg1 did not cause a substantial GFP+% and MFI decrease.e– fTargeted deep sequencing analysis of indel frequencies at rDNA loci caused by the endonuclease activity of R2 retrotransposons (eor Cas9 (f. Indel frequencies were specified as the percentage of rDNA loci including indels relative to the overall rDNA swimming pool, stabilized by deducting background worths from unfavorable controls.gSorted GFP+ cells (from Extended Data Fig. 8a) sustain steady and high-efficiency transgene expression throughout long-lasting culture.h-iTransfection of R2 protein-encoding mRNA into stably GFP-expressing cells (originated from Extended Data Fig. 8 g) caused indel development (
aria-labelledby=19459012 data-title=19459013> References 19460578
- Villiger, L. et al. CRISPR innovations for genome, epigenome and transcriptome modifying . Nat. Rev. Mol. Cell Biol. 19460598 19460599
- 19460585 19460602 Authors and Affiliations 19460888 19460579 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 19460579 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 19460585 19460602 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China 19460585 Yangcan Chen, Yanping Hu, Na Tang & , Chenxin Wang, Xinge Wang, Peng Wang, Ng Shyh-Chang, Tuo Wei, Haoyi Wang, Qi Zhou & Wei Li 19460585 19460602 19460579 Authors 19460585 Yangcan Chen 19460887 19460896 Yanping Hu 19460887 Shengqiu Luo 19460887 19460896 Bangwei Mao 19460602 Na Tang 19460602 Chenxin Wang 19460887 19460896 Yuze Wang 19460887 Hanyu Bai Xinge Wang 19460887 19460896 Changxian Peng 19460887 19460602 Qicheng Chen 19460887 19460602 19460896 Jiahui Xiao 19460602 Yu Zou 19460602 19460896 Peng Wang Rui Niu 19460602 19460896 Jin Zhang 19460602 19460896 Yulong Zhao Chen Liang Yunlong Wang 19460887 Yiyun Li 19460887 19460896 Junjie Wang 19460887 Ng Shyh-Chang 19460602 19460896 Tuo Wei 19460887 19460602 Haoyi Wang 19460887 19460602 19460896 Qi Zhou 19460896 Wei Li 19460602 19460586 Contributions W.L., Q.Z. and Y.C. developed the task and created 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. carried out the experiments. W.L., Q.Z., Y.C., Y.H. and S.L. evaluated the information. W.L., Q.Z., Y.C. and H.W. monitored the research study. W.L., Q.Z., Y.C., Y.H., S.L., N.S.-C., T.W. and H.W. composed the paper, helped by the other authors. 19460585 Corresponding authors 19460891 Correspondence to Yangcan Chen, Haoyi Wang, Qi Zhou or Wei Li. 19460586
Ethics statements 19460578 Contending interests 19460891 W.L., Q.Z., Y.C., Y.H., S.L., Q.C., X.W. and B.M. have actually sent patent applications associated to this paper. The other authors state no contending interests. 19460586 19460586
Peer evaluation Peer evaluation info 19460579 19460597 Nature Biotechnology 19460598 thanks Lei Qi, Max Wilkinson and the other, confidential, customer(s )for their contribution to the peer evaluation of this work.
19460586 Additional info 19460578 Publisher’s note Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19460585
Extended information 19460940 Extended Data Fig. 1 Workflow for mining brand-new R2 retrotransposons in bird genomes. 19460891 19460599 a , Computational pipeline for determining bird R2 retrotransposons. 19460599 b , Distribution of freshly found R2 aspects throughout orders of evaluated bird genomes. 19460599 c , Left: circulation of downloaded bird genomes throughout various orders; Right: circulation of bird genomes consisting of full-length R2 components throughout various orders. 19460585 Extended Data Fig. 2 Systematic characterization of Region-1 and Region-2 in the R2Tg protein. 19460579 a 19460600, Droplet digital PCR (ddPCR)analysis shows that Region-1 truncation considerably hinders the molecular combination effectiveness of R2Tg, and this flaw can be saved by HMGN1 blend. Cp, copies. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. b 19460600, Effect of Region-1 truncation length on R2-mediated gene combination performance in HEK293T cells, paired with donor 19460583 choose 19460584 c 19460600, Identification of replacing domains capable for saving Region-1 function in HEK293T cells, combined with donor choose NLS1/2, Nuclear Localization Signal 1/2 17 ; NBD, nucleosome-binding domain; CHUD, chromatin unfolding domain. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 d , Trans-expression of Region-1 stops working to save R2-mediated gene combination effectiveness in HEK293T cells, combined with donor choose 19460584 Unpaired, two-tailed Student’s t tests. e 19460600, Impact of region-2 truncation on R2-mediated gene combination activity in HEK293T cells, combined with donor decide 19460584 Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test. 19460599 f , Statistical analysis of Region-1 length throughout 165 bird R2 retrotransposon proteins. 19460599 g , Statistical analysis of length of unique subregions (Spacer, NTE-2 and partial NTE-1) in Region-2 throughout 165 bird R2 retrotransposon proteins. h 19460600, Evaluating the activity of R2Tg protein with gotten rid of nuclear localization signal or rearranged Flag tag in all-RNA speculative systems in HEK293T cells, paired with donor decide 19460584 Unless otherwise mentioned, information exist as mean ± basic variance (SD) from 3 biological reproduces. 19460585 Extended Data Fig. 3 Investigation of the impacts of donor RHA length and polyA length on R2-mediated gene combination effectiveness. 19460891 19460579 a , Impact of donor RNA with differing best homology arm (RHA)and polyA lengths on combination performance in HEK293T cells. United States, unassociated vector series. 19460599 b 19460600, Impact of donor RNA with 4 nt RHA and different polyA lengths in HEK293T cells. c 19460600, Integration performance of R2Tg v1.1 19460584 with donor RNAs including various nucleotide elements after a 4 bp RHA in HEK293T cells. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates. 19460585 19460940 Extended Data Fig. 4 Investigation of the results of 5’UTR components on R2-mediated gene combination performance. 19460891 a , Statistical analysis of J1/2 area length throughout 165 bird R2 5’ribozymes. 19460599 b 19460600, Predicted secondary structure of the J1/2 area in 165 bird R2 5 ‘ribozymes, presumed through covariation modeling. 19460599 c 19460600, Statistical analysis of the length of non-ribozyme part in 165 R2 5’UTRs. d , Predicted secondary structure of the non-ribozyme part in bird R2 5’UTRs from Aves_c1 to c5, presumed through covariation modeling. 19460599 e , Integration performance of R2Tg 19460583 v1.1 with donor RNAs harboring customized 5′ UTR series in HEK293T cells. Sub1– 10 represents replacement of the very first 1– 10 nt of the 5′ UTR with heterologous series to interrupt the J1/2 secondary structure. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s numerous contrasts test. 19460599 f , Length circulation of the buffer area in the bird 3′ UTR. g 19460600, Nucleotide structure of the buffer area in the bird 3’ UTR. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460586 19460940 Extended Data Fig. 5 Systematic contrast of the R2 tool style method established in this research study with those of formerly reported R2 tools. 19460891 19460599 a 19460600, Summary of the styles of crafted R2 retrotransposons in this research study and 2 previous research studies (Study 1 19460583 7 and Study 2 6. Mini-donor from study2 was utilized for contrast. St1, study1; St2, study2. 19460599 b , Comparison of gene combination activity of R2 versions from various styles, with cross-combination recognition of R2 proteins and donor RNAs in HEK293T cells. mRNAs encoding R2Tg shared similar codon use, translational UTRs and poly(A) tail lengths; donor RNAs were built as explained in the initial reports, including their native promoters, terminators and transgene expression cassette orientations. c 19460600, Schematic diagram of donor RNAs with differing lengths. d-e 19460600, Comparison of the gene combination capability of R2 tools from various styles utilizing RNA donors with lengthened stuffer series ( 19460599 d 19460600 or RNA donors bring gene-T2A-GFP expression cassettes ( 19460599 e 19460600 in HEK293T cells. f 19460600, Schematic of the RNA donors with dual-fluorescence press reporter system (GFP and mCherry) for various R2 tools. g 19460600, Gating technique for circulation cytometric analysis of double fluorescence. 19460599 h , Assessment of the dual-fluorescence gene combination effectiveness of R2 tools from various styles. 19460599 i 19460600, Comparison of the stability of gene combination items moderated by R2 tools from various styles. Analytical analyses were carried out utilizing one-way ANOVA with Tukey’s several contrasts test. For all charts, information are represented as mean ± SD from 3 biological reproduces. 19460585
19460940 Extended Data Fig. 6 Investigation of the elements affecting differential activity amongst R2 tools styles. a , Replacement of the 5′ UTR in R2Tg donor vx 19460584 with that originated from 19460597 Tribolium castaneum 19460598 considerably decreased the gene combination activity in HEK293T cells. Unpaired, two-tailed Student’s t test. 19460599 b 19460600, Evaluation of combination effectiveness moderated by R2Tg v1.1 , where donor 19460583 vx brought 28-nt, 36-nt, or 100-nt left homology arms (LHA) with or without the 5′ UTR and 5′ Cap-1 in various speculative groups in HEK293T cells. c , Analysis of combination performance moderated by R2Tg v1.1 with donors harboring various transgene expression constructs (all-RNA system) in HEK293T cells. Reverse constructs: expression cassette opposite to rDNA transcription instructions; Forward constructs: cassette lined up with rDNA transcription instructions. 1– 9 represent various promoter-polyA terminator mixes. MFI: indicate fluorescence strength of GFP + cells. 19460599 d , ddPCR metrology of gene combination copy numbers representing the unique RNA donor constructs in Panel C.eCorrelation analysis in between transgene copy number and fluorescence strength for all constructs in Panel C by means of direct regression. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP +cells, showing the relative overall fluorescence strength of each sample.fCorrelation analysis in between transgene copy number and fluorescence strength for CMV-containing constructs in Panel C through direct regression.gPercentage and MFI of GFP +cells after transfection of matching IVT-donor plasmids utilizing Lipofectamine 3000 in HEK293T cells. For all charts, information are represented as mean ± SD from 3 biological reproduces.Extended Data Fig. 7 Systematic characterization of the R2 tools in regards to transgene copy number, combination uniqueness and full-length insertion capability in HEK293T cells.
aR2 transgene combination copy numbers in the human genome, measured by means of ddPCR. Unpaired, two-tailed Student’s t tests.bLinear regression analysis of overall fluorescence strength versus matching transgene copy numbers. Fluorescence strength of each sample was determined as MFI increased by the portion of GFP+cells, showing the relative overall fluorescence strength of each sample.cWhole-genome sequencing revealing high on-target combination uniqueness of R2 retrotransposons in mammalian cells.dAssessment of the stability of R2-integrated transgene by means of whole-genome sequencing.e– jInvestigation of elements affecting the full-length transgene insertion percentage of the R2 retrotransposon tool utilizing the GFP-T2A-mCherry dual-fluorescence press reporter system;(e)Portion of fluorescence-positive cells and(f)percentage of full-length transgene insertions (examining results of donor styles). FC, forward construct. Analytical analyses were carried out utilizing one-way ANOVA with Dunnett’s several contrasts test.(g)Portion of fluorescence-positive cells and(h)percentage of full-length transgene insertions (evaluating results of R2Tg protein styles). Unpaired, two-tailed Student’s t tests.(i)Portion of fluorescence-positive cells and(j)percentage of full-length transgene insertions (evaluating results of shipment reagents). Unpaired, two-tailed Student’s t tests. Unless otherwise mentioned, information exist as mean ± basic discrepancy (SD) from 3 biological duplicates.
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Acknowledgements 19460578 19460579 We want to thank J. Sun for assistance on T cell control. We likewise thank the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, for offering the main HHFs utilized in this research study. 19460586
Funding 19460578 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. 19460585 19460586
Author info 19460578 Author notes 19460887 19460888 19460579 These authors contributed similarly: Yangcan Chen, Yanping Hu, Shengqiu Luo, Bangwei Mao, Na Tang, Chenxin Wang, Yuze Wang.
Extended Data Fig. 8 Long-term tracking of transgene expression moderated by R2 combination in HEK293T cells.
aPercentage(left)and MFI(ideal)of GFP+cells throughout long-lasting constant culture after R2-mediated combination.bCopy varieties of incorporated transgenes throughout long-lasting culture( from Extended Data Fig. 8a). Paired, two-tailed Student’s t test.cMixed culture of GFP+cells(from Extended Data Fig. 8a )and unfavorable cells(1:1 ratio) reveals faster decrease in GFP+portion than forecasted. Anticipated GFP+portion: half the favorable worth from Extended Data Fig. 8a at matching days; anticipated MFI: MFI of GFP+cells from Extended Data Fig. 8a.dCRISPR-Cas9-induced rDNA recombination minimizes the portion and MFI of GFP+cells; especially, nCas9(H840A)+sg1 did not cause a substantial GFP+% and MFI decrease.e– fTargeted deep sequencing analysis of indel frequencies at rDNA loci caused by the endonuclease activity of R2 retrotransposons (eor Cas9 (f. Indel frequencies were specified as the percentage of rDNA loci including indels relative to the overall rDNA swimming pool, stabilized by deducting background worths from unfavorable controls.gSorted GFP+ cells (from Extended Data Fig. 8a) sustain steady and high-efficiency transgene expression throughout long-lasting culture.h-iTransfection of R2 protein-encoding mRNA into stably GFP-expressing cells (originated from Extended Data Fig. 8 g) caused indel development (
Extended Data Fig. 8 Long-term tracking of transgene expression moderated by R2 combination in HEK293T cells.
aPercentage(left)and MFI(ideal)of GFP+cells throughout long-lasting constant culture after R2-mediated combination.bCopy varieties of incorporated transgenes throughout long-lasting culture( from Extended Data Fig. 8a). Paired, two-tailed Student’s t test.cMixed culture of GFP+cells(from Extended Data Fig. 8a )and unfavorable cells(1:1 ratio) reveals faster decrease in GFP+portion than forecasted. Anticipated GFP+portion: half the favorable worth from Extended Data Fig. 8a at matching days; anticipated MFI: MFI of GFP+cells from Extended Data Fig. 8a.dCRISPR-Cas9-induced rDNA recombination minimizes the portion and MFI of GFP+cells; especially, nCas9(H840A)+sg1 did not cause a substantial GFP+% and MFI decrease.e– fTargeted deep sequencing analysis of indel frequencies at rDNA loci caused by the endonuclease activity of R2 retrotransposons (eor Cas9 (f. Indel frequencies were specified as the percentage of rDNA loci including indels relative to the overall rDNA swimming pool, stabilized by deducting background worths from unfavorable controls.gSorted GFP+ cells (from Extended Data Fig. 8a) sustain steady and high-efficiency transgene expression throughout long-lasting culture.h-iTransfection of R2 protein-encoding mRNA into stably GFP-expressing cells (originated from Extended Data Fig. 8 g) caused indel development (
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