Abstract
Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.
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Data availability
All high-throughput sequencing data (ATAC-seq, Hi-C, ChIP–seq, RNA-seq, whole-genome sequencing and whole-genome bisulfite sequencing) in this paper are contained in the Sequence Read Archive (SRA) (PRJNA970280). The raw gel image of PCR, the raw data of flow cytometry and the data of stress treatments are provided in the Supplementary Information. The BigWig files of ChIP–seq are available at figshare (https://figshare.com/articles/dataset/ChIP_track_rar/23648046). P. patens genome v.3.3 is available at Phytozome (https://phytozome-next.jgi.doe.gov). Source data are provided with this paper.
Code availability
All original codes used in high-throughput sequencing analysis have been deposited at Github (https://github.com/lanntianlong/SynMoss) and Zenodo (https://doi.org/10.5281/zenodo.8000393). The inhouse computational pipeline for standardizing genome design is available on Zenodo (https://doi.org/10.5281/zenodo.7894207).
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Acknowledgements
This work was funded by the National Key R&D Program of China grant no. 2019YFA0903900 (Q.Z., W.Y., W.Q. and Y.J.), the National Natural Science Foundation of China grant nos. 31825002 (Y.J.), 31800069 (J.D.), 31800082 (J.D.) and 32270345 (Y.W.), Shenzhen Science and Technology Program grant no. KQTD20180413181837372 (J.D.), Guangdong Provincial Key Laboratory of Synthetic Genomics grant no. 2019B030301006 (J.D.), Guangdong Basic and Applied Basic Research Foundation grant no. 2023A1515030285 (J.D.), Bureau of International Cooperation, Chinese Academy of Sciences grant no. 172644KYSB20180022 (J.D.), CAS Strategic Priority Research Program grant nos. XDA24020203 (Y.J.), XDA24020103 (W.Q.) and XDA28030402-5 (W.Q.), Shenzhen Outstanding Talents Training Fund (J.D.) and the China Postdoctoral Science Foundation no. 2020M680740 (L.-G.C.). We thank the National Center for Protein Sciences at Peking University for assistance with flow cytometry analysis.
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Authors and Affiliations
Contributions
Y.J., J.D., W.Q., Y.W., L.-G.C. and Q.Z. designed the experiments and analysed the data. S.Z., Y.Z., Q.D., B.J. and W.Q. performed genome design. M.Z., G.L., Z.H and Y.M. performed genome assembly in yeast. L.-G.C. and T.L. performed plant genome assembly, genome sequencing, transcriptome, epigenome and Hi-C analyses. L.-G.C., T.L., H.L. and B.L. performed genotyping and sequence analysis. Y.G. performed phenotype analysis. Y.J., J.D., W.Q., Y.W., L.-G.C., T.L. and S.Z. wrote the paper.
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L.-G.C., Y.W. and Y.J. are inventors on patent applications covering the results described in this study. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 The design and assembly of three 20–30 kb mid-chunks.
We split the 68,530 bp mega-chunk into three mid-chunk fragments and added a resistant cassette (kanamycin or hygromycin) to the centromeric end of each mid-chunk. If it is not possible to achieve the goal of replacing three 30 kb mid-chunks in the assembly, the replacement of two or one mid-chunks should be implemented. Each mid-chunk overlapped with the next one by a 1 kb overhang and 1 kb homologous arms were included at either end of the entire region.
Extended Data Fig. 2 Characterization of syn-50k.
(a) PCRTag analysis. A total of ten recombination replacement sites were detected, including eight sites in deleted regions and two in homologous arms. Only synthetic sequences and not wild-type sequences can be amplified from semi-syn18L. Three independent experiments were conducted on each line using three independent samples and similar results were obtained. (b) Whole-genome Illumina resequencing analysis of syn-50k. No reads containing sequences from the deleted regions were found and reads were identified covering all new junctions. (c) Flow cytometry analysis indicated that semi-syn18L-2 was as haploid as the wild type. (d) The phenotypes of the wild type and syn-50k at various developmental stages. The figure shows the phenotypes of representative samples. (More than 3 independent samples were observed for each stage and each line). Bars from left to right, 500 μm, 50 μm, 50 μm, 20 μm, 2 mm, 1 mm and 500 μm.
Extended Data Fig. 3 Schematic diagram of the replacement of the synthetic region in 7 partial replacement lines.
The light yellow segment represents the region that should have been deleted according to the design and the green segment represents the preserved region.
Extended Data Fig. 4 Stress treatment of wild type and semi-syn18L.
(a) NaCl treatment. The chlorophyll content of wild type and semi-syn18L was determined after 0 mM, 200 mM, 300 mM, 400 mM and 500 mM NaCl stress treatments (3 d). The bars shown in the histogram are means ± SDs. The points in the figure show the specific values in each repetition, n= 3 biological replicates. (b) Sorbitol treatment. The chlorophyll contents of wild type and semi-syn18L were determined after 0 mM, 400 mM, 500 mM, 600 mM and 750 mM sorbitol stress treatments (3 d). The bars shown in the histogram are means ± SDs. The points in the figure show the specific values in each repetition, n = 3 biological replicates.
Extended Data Fig. 5 Heatmap showing correlations among histone marks in the wild type and semi-syn18L.
The correlation coefficient was calculated using the normalized read counts in each peak region. Colours represent the correlation coefficients, with darker green indicating higher similarity. Hierarchical clustering is shown at the left and top of the heatmap. Note that active histone modifications cluster together, while H3K9me2, which often represents heterochromatin, differs the most from other modifications. Overall, there is minimal difference in the distribution of histone modifications between the wild type and semi-syn18L from a genome-wide perspective. (also see Supplementary Table 7).
Extended Data Fig. 6 Other epigenetic changes in semi-syn18L.
(a) Distribution of five histone modifications (H3K4me3, H3K9me2, H3K27me3, H3K36me3 and H3K27ac) on genes in the synthetic region. The signals shown were normalized using the BPM method (Bins Per Million mapped reads, same as TPM in RNA-seq) after deducting the input. (b) The number of peaks for histone modifications in wild-type and semi-syn18L. The left figure shows the number of histone modification peaks across the entire genome, while the right figure shows the number of histone modification peaks in the replacement region. The counts of three replicates were separately calculated. (c) Changes in H3K9me2 modification and DNA methylation levels at the remaining intergenenic region in semi-syn18L. The top panel shows designed chromosome fragment. The Xs on the top panel refer to new joints formed by deleting duplicate sequences, which correspond to the yellow delete regions below.
Supplementary information
Supplementary Information
Supplementary Box 1 and Tables 1–6, 8 and 9.
Supplementary Table 1
Notable changes in histone modification peaks and ATAC peaks detected by DiffBind.
Supplementary Data 1
The wild-type genome sequence for the phase I designs.
Supplementary Data 2
Corresponding annotations of the wild-type genome sequence for the phase I designs.
Supplementary Data 3
The wild-type genome sequence for the phase II designs.
Supplementary Data 4
Corresponding annotations of the wild-type genome sequence for the phase II designs.
Supplementary Data 5
The simplified genomes in phase I.
Supplementary Data 6
Corresponding annotations of the simplified genomes in phase I.
Supplementary Data 7
The simplified genomes of phase II.
Supplementary Data 8
Corresponding annotations of the simplified genomes in phase II.
Source data
Source Data Fig. 1
Unprocessed gels.
Source Data Fig. 2, 4 and Source Data Extended Data Fig. 2
Microscope image.
Source Data Fig. 2, 4 and Source Data Extended Data Fig. 2
Raw results of flow cytometry.
Source Data Fig. 4
Unprocessed gels.
Source Data Fig. 4
Microscope image.
Source Data Extended Data Fig. 2
Unprocessed gels.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 6
Statistical source data.
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Chen, LG., Lan, T., Zhang, S. et al. A designer synthetic chromosome fragment functions in moss. Nat. Plants 10, 228–239 (2024). https://doi.org/10.1038/s41477-023-01595-7
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DOI: https://doi.org/10.1038/s41477-023-01595-7
