CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci

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CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci

Data availability

The data supporting this study are provided in the article and in its Supplementary Information and Source Data files. Multi-omics data from published studies used in this work are summarized in Supplementary Table 3. Additional data are available at https://www.wanglab-thu.org.cn/pro. Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the Wang laboratory for their discussions and comments during the preparation of the manuscript. We thank the Imaging Core Facility of the Technology Center for Protein Sciences at Tsinghua University for microscopy imaging and analysis support. We thank R. Xu and D. S. Liu for their support in exploring fluorescent gRNA labeling methods during the initial stages, which, although not included here, has provided valuable insights. This work was funded by the following grants: the Agriculture Science and Technology Major Project to H.W., the National Key R&D Program of China (2022YFC3400204 to H.W., 2021YFA1301500 to X.F., 2021YFA1100102 to W.X.); the National Natural Science Foundation of China (32270573 to H.W., U1832215 to X.F.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0570300 to X.F.), the Beijing Natural Science Foundation (F251024 to X.F.) and the Tsinghua-Peking Joint Center for Life Sciences (H.W. and W.X.). H.W. is supported by the Independent Research Fund of the State Key Laboratory of Complex, Severe, and Rare Diseases (2025-I-ZD-005), the Tsinghua University Initiative Scientific Research Program and the Benyuan Charity Fund. W.X. is a recipient of the New Cornerstone Investigator award.

Author information

Author notes

  1. Zhenhai Du

    Present address: State Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

  2. These authors contributed equally: Meishuo Liu, Keyun Huang, Jie Zhang.

Authors and Affiliations

  1. School of Life Sciences, Tsinghua-Peking Joint Center for Life Sciences, Center for Synthetic and Systems Biology, State Key Laboratory of Complex, Severe, and Rare Diseases, Tsinghua University, Beijing, China

    Meishuo Liu, Keyun Huang, Qingyang Li, Xinming Wang, Buming Gu, Hao Tang, Yu Ma & Haifeng Wang

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Jie Zhang

  3. State Key Laboratory of RNA Innovation, Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jie Zhang & Xianyang Fang

  4. Center for Stem Cell Biology and Regenerative Medicine, MOE Key Laboratory of Bioinformatics, New Cornerstone Science Laboratory, School of Life Sciences, Tsinghua University, Beijing, China

    Zhenhai Du, Liangjun Hu & Wei Xie

  5. Tsinghua-Peking Joint Center for Life Sciences, Beijing, China

    Zhenhai Du, Liangjun Hu & Wei Xie

  6. New Cornerstone Science Laboratory, School of Life Sciences, Westlake University, Hangzhou, China

    Shutao Qi & Hongtao Yu

  7. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Shutao Qi & Hongtao Yu

Authors

  1. Meishuo Liu
  2. Keyun Huang
  3. Jie Zhang
  4. Qingyang Li
  5. Xinming Wang
  6. Buming Gu
  7. Hao Tang
  8. Zhenhai Du
  9. Liangjun Hu
  10. Shutao Qi
  11. Yu Ma
  12. Hongtao Yu
  13. Wei Xie
  14. Xianyang Fang
  15. Haifeng Wang

Contributions

H.W., M.L. and K.H. conceived and designed the study. H.W., W.X., X.F. and H.Y. supervised the work. M.L. and K.H. performed the main experiments, with the help of H.T., Z.D., L.H., S.Q. and Y.M. K.H., J.Z. and Q.L. collaborated on the fluorescent labeling of sgRNAs. B.G., X.W., M.L., K.H., Q.L. and H.T. analyzed the data. H.W., M.L. and K.H. wrote the manuscript, with the help of X.F., W.X. and H.Y.

Corresponding authors

Correspondence to Wei Xie, Xianyang Fang or Haifeng Wang.

Ethics declarations

Competing interests

The authors have filed patents (CN: 202511377180X by H.W., X.F., K.H., J.Z. and M.L. and CN: 2025113774687 by H.W., M.L. and K.H.) related to this work.

Peer review

Peer review information

Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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Extended data

Extended Data Fig. 1 Additional LiveFISH imaging of specific gene loci via fluorescent crRNAs or fluorescent tracrRNAs.

a, Additional live-cell images of various repetitive loci, including Chr3, MUC4, FBN3, and ZNF34, labeled by crRNA-LiveFISH. Scale bars, 5 μm. b, Comparison of signal-to-noise ratio (SNR) at gene loci with different numbers of repeats, including Chr3 (~500 copies, N=100 loci, Cy3), MUC4 (82 copies, N=100 loci, Atto565), FBN3 (22 copies, N=100 loci, Atto565), and ZNF34 (20 copies, N=100 loci, Atto565). Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points. c, Additional live-cell images of Chr3 and MUC4, labeled by tracr-LiveFISH. Scale bars, 5 μm. d, A representative U2OS image (left) of MUC4 loci using a pair of crRNA (red, Atto565) and tracrRNA (green, Cy5) labeled with distinct fluorophores. Scale bar, 5 μm. e, The linescan plot showing signal-to-background ratio along the dashed line in d. f, A representative U2OS image of ZNF34 loci using a pair of crRNA (red, Atto565) and tracrRNA (green, Cy5) labeled with distinct fluorophores. Scale bar, 5 μm. g, The linescan plot showing signal-to-background ratio along the dashed line in f. h, Comparison of SNR at ZNF34 loci by fluorescent crRNA (N=100 loci, Atto565) and fluorescent tracrRNA (N=100 loci, Cy5). p-value: two-sided unpaired t-test. Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points. All experiments were replicated independently ≥ 3 times.

Extended Data Fig. 2 Additional information for validating the labeling efficiency and specificity of UBP-sgRNA-based LiveFISH imaging.

a, Schematic of CRISPR LiveFISH via randomly inserted Cy3-labeled sgRNA for repetitive sequences. The DNA template was prepared by PCR using a forward primer containing T7 promoter and a spacer sequence, and a universal reverse primer containing the sgRNA scaffold. Fluorescent sgRNAs were prepared by random insertion of Cy3-UTP during IVT, assembled with dCas9, and delivered into living cells for LiveFISH imaging. b, 8% TBE denaturing urea PAGE analysis of Cy3-labeled sgRNAs generated by UBP labeling (lanes 1–3) and random-insertion labeling (lanes 4). Visualized by fluorescent imaging (left) and SYBR gold staining (UV, right). A single-stranded RNA ladder was used to verify the sgRNAs targeting Chr3, MUC4, and ZNF34 with different spacer lengths. c, A representative U2OS image of Chr3 loci using Cy3-RI-sgRNA. Scale bar, 5 μm. d, Additional live-cell images of MUC4 and ZNF34 labeled by UBP-based LiveFISH. Scale bars, 5 μm. e, DNA FISH (green) validating UBP-sgRNA LiveFISH (red) labeling of ZNF34 loci, with separate channels. Scale bar, 5 μm. f, RNA FISH (green) validating UBP-sgRNA labeling (red) of ZNF34 loci. Scale bar, 5 μm. g, Relative ZNF34 expression in U2OS cells after delivery of ZNF34-targeting sgRNA (sgTarget) versus non-targeting sgRNA (sgNT), normalized to the non-targeting group. Mean ± SD. Individual points show data from 3 biological replicates. p-value: two-sided unpaired t-test. h, Representative U2OS cell imaging of MUC4 loci using UBP-sgRNA (JF549) with dCas9-EGFP. Scale bar, 5 μm. i, The linescan plots showing relative fluorescence intensity of UBP-sgRNA and dCas9-EGFP along the dashed lines in h. j, Comparison of SNR between dCas9-EGFP (N=100 loci) and UBP-sgRNA (N=100 loci, JF549) at MUC4 loci. Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points; p-value: two-sided unpaired t-test. All experiments were replicated independently ≥ 3 times.

Source data

Extended Data Fig. 3 Additional information on multi-color imaging using UBP-sgRNA-based LiveFISH.

a, Additional U2OS cells showing dual-color labeling of the MUC4 (red, AF647) and ZNF34 loci (green, JF549) using UBP-sgRNAs, respectively. Scale bars, 5 μm. b, Live-cell imaging revealed fluorescent dCas9 exchange between different pre-assembled RNPs. Two sets of fRNPs were separately pre-assembled: dCas9-EGFP (green) with JF549-sgRNAChr13 (blue), and dCas9 with AF647-sgRNAChr3 (red). These RNPs were then co-delivered into U2OS cells at a ratio of 1:1. If no crosstalk occurs, Chr3 loci would be labeled exclusively by AF647-sgRNAChr3 (red). However, due to RNP crosstalk, Chr3 loci were labeled in both the red (AF647-sgRNAChr3) and green (dCas9-EGFP) channels, indicating that dCas9-EGFP exchanged between the two pre-assembled RNP complexes (scale bar, 5 μm; insets, scale bars, 1 μm). c, A representative U2OS cell showing simultaneous labeling of six loci using differentially labeled UBP-sgRNAs (scale bars, 5 μm) with insets (scale bars, 1 μm), including T1 (AF488), FBN3 (JF549), ZNF34 (AF647), Chr13 (AF488 and JF549), MUC4 (JF549 and AF647) and PR1 (AF488 and AF647). d, The 3D trajectories of the non-repetitive MUC4.I1 loci (red), MUC4.I3 loci (green), and Chr3 loci (purple) in Fig. 3c. e, Additional live-cell images labeling the non-repetitive MUC4.I1 loci (red, JF549), repetitive MUC4.I3 loci (green, AF647), and Chr3 loci (purple, AF488; scale bars, 5 μm). Insets: scale bars, 1 μm. f, Box plot comparing the distances from MUC4.I1 to MUC4.I3 (N=100 loci pairs), and from Chr3 to MUC4.I3 (N=100 loci pairs), respectively. Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points; p-value: two-sided unpaired t-test. All experiments were replicated independently ≥ 3 times.

Extended Data Fig. 4 Additional epigenetic characterization, gRNA design, FISH imaging, and RNA expression analysis of the Xist locus.

a-c, Bar graphs of the ATAC-seq (a), H3K4me1 (b), and H3K27me3 (c) signals around the Xist gene in NIH3T3 (blue), MEF (grey), and XEN (orange) cells. d, Schematic of the Xist gene labeled by PRO-LiveFISH using a pool of fluorescent gRNAs. e, Additional live-cell images labeling the Xist loci in NIH3T3 (male), MEF (female), and XEN (female) cells by PRO-LiveFISH. Scale bars, 5 μm. f, Representative XEN cell imaging of the Xist DNA locus by PRO-LiveFISH (red) and Xist RNA transcripts (grey, RNA FISH) around the inactive X chromosome. Scale bar, 5 μm. g, Relative Xist expression in XEN cells after delivery of Xist-targeting sgRNA (sgTarget) or non-target sgRNA (sgNT), normalized to the non-targeting group. Mean ± SD. Individual points show data from 3 biological replicates. p-value: two-sided unpaired t-test. h, Box plot comparing the sizes of Xist loci in NIH3T3 (blue, N=11 loci), MEF (grey, N=11 loci), and XEN (orange, N=11 loci) cells, respectively. p-value: one-way ANOVA with Tukey’s post hoc test. i, Representative DNA FISH images of the Xist loci in the XEN cell. Scale bar, 5 μm. j, Comparison of the sizes of Xist loci in XEN cells labeled by PRO-LiveFISH (N=14 loci) and DNA FISH (N=14 loci). p-value: two-sided unpaired t-test. k, Scatterplots for Xist step displacement (dx, dy) in XEN cells, including control group (Ctrl, DMSO treated) and A-485 treated group (A485, 100 μM for 16 h). dxt = xt-xt-1, dyt = yt-yt-1, where (xt,yt) is the locus coordinate at time t. Movements tracked every 5 s. Box plots in h and j show median, 25th–75th percentiles, whiskers (10th–90th), and individual points. All experiments were replicated independently ≥ 3 times.

Extended Data Fig. 5 Additional information on PRO-LiveFISH and DNA FISH imaging of PCDHα-enhancer interactions.

a, Additional U2OS cell imaging showing PCDHα (red, AF647) and HS5-1 loci (green, JF549; scale bar, 5 μm), with magnified insets showing time-lapse images with split channels (bottom; scale bars, 1 μm). b, Representative DNA FISH image of the PCDHα (green) and HS5-1 (red) loci in U2OS cell. Scale bar, 5 μm. c, Quantification of distances between PCDHα and HS5-1 by DNA FISH in U2OS cells. Mean ± SD, with dots showing 3 independent biological replicates. d, Comparison of distances between PCDHα and HS5-1 by PRO-LiveFISH (N=150 loci pairs) and DNA FISH (N=120 loci pairs). Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points; p-value: two-sided unpaired t-test. All experiments were replicated independently ≥ 3 times.

Extended Data Fig. 6 Additional information on PRO-LiveFISH imaging of Xist gene and its enhancer in primary MEF cells.

a, Imaging of a representative female primary MEF cell labeling the Xist loci by PRO-LiveFISH, based on the pooled JF549-sgRNAs. Scale bar, 5 μm. b, Imaging of a representative male primary MEF cell labeling the Xist loci by PRO-LiveFISH, based on the pooled JF549-sgRNAs. Scale bar, 5 μm. c, DNA FISH images of the Xist loci in female primary MEF cells. Scale bar, 5 μm. d, DNA FISH images of the Xist loci in male primary MEF cells. Scale bar, 5 μm. e, The integrated map of Hi-C and H3K27ac signals (ChIP-seq) for the Xist and Ftx genomic regions in MEF cells. f, DNA FISH images of the Xist and Ftx loci in the female (top) and male (bottom) primary MEF cells. Scale bars, 5 μm. g, Comparison of distances between Xist and Ftx loci by PRO-LiveFISH (N=100 loci pairs) and DNA FISH (N=100 loci pairs) in primary MEF cells. Box plots show median, 25th–75th percentiles, whiskers (10th–90th), and individual points; p-value: two-sided unpaired t-test. All experiments were replicated independently ≥ 3 times.

Extended Data Fig. 7 Additional information on studying BRD4’s role in super-enhancer organization of the MYC oncogene.

a, Magnified time-lapse and split-channel images of inset 1 in Fig. 6b. Scale bars, 1 μm. b, Additional live-cell images of MYC (green, JF549) and its SE (CCAT1, red, AF647) in HeLa cells using PRO-LiveFISH (scale bars, 5 μm), with insets (scale bars, 1 μm). c, Representative HeLa cells showing labeling of MYC SE (CCAT1) by PRO-LiveFISH and MYC RNA (RNA FISH). Scale bars, 5 μm. d, The relative MYC expression in HeLa cells after delivery of CCAT1-targeting sgRNA (sgTarget) or non-target sgRNA (sgNT), normalized to the non-targeting group. Mean ± SD. Individual points show data from 3 biological replicates. e, Magnified time-lapse and split-channel images of inset 1 in Fig. 6e. Scale bars, 1 μm. f, Additional live-cell images in HeLa cells after JQ1 treatment labeling MYC (green, JF549) and its SE (CCAT1, red, AF647) using PRO-LiveFISH (scale bars, 5 μm), with insets (scale bars, 1 μm). g, Distribution of distances between MYC and its SE (CCAT1) in control (Ctrl, DMSO) and JQ1-treated group (5 μM, 24 h). Mean ± SD, with dots showing 3 independent replicates. h, Box plot of distances between MYC and its SE (CCAT1) in control (Ctrl, N=120 loci pairs) and JQ1-treated group (N=120 loci pairs) by DNA FISH. i, Representative DNA FISH images of the MYC and its SE (CCAT1) in control (Ctrl, top) and JQ1-treated group (scale bars, 5 μm), with insets (scale bars, 1 μm). j-k, Box plots of 3D step distances of MYC (j) and CCAT1 (k) in control (Ctrl, N=150 loci steps) and JQ1-treated groups (JQ1, N=150 loci steps), tracked at 10-s intervals. p-value in d, h, j and k: two-sided unpaired t-test. Box plots in h, j and k show median, 25th–75th percentiles, whiskers (10th–90th), and individual points. All experiments were replicated independently ≥ 3 times.

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Liu, M., Huang, K., Zhang, J. et al. CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02887-3

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