Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing


CRISPR–Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR–Cas9-mediated homology-independent organoid transgenesis (CRISPR–HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR–HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR–HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter—in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin—uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR–HOT simplifies genome editing in human organoids.

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Fig. 1: CRISPR–HOT strategy and efficiency.
Fig. 2: CRISPR–HOT enables precise gene knock-in.
Fig. 3: Generation of human liver ductal reporter organoid lines using CRISPR–HOT.
Fig. 4: Labelling of different human intestinal cell types in organoids using CRISPR–HOT.
Fig. 5: Generation of human hepatocyte reporter organoid lines using CRISPR–HOT.
Fig. 6: Mitotic spindle analyses in human hepatocyte reporter organoids reveal division dynamics.
Fig. 7: Loss of TP53 causes mitotic spindle aberrations in human hepatocytes.

Data availability

Plasmids generated in this study (Extended Data Fig. 1) are deposited in Addgene (138567, 138568, 138569, 138570 and 138571). Genotyping data that support the findings of this study have been deposited in GenBank under the following accession codes: MN952225, MN952226, MN952227, MN952228, MN952229, MN952230, MN952231 and MN952232. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data for Figs. 1, 2, 6 and 7, and Extended Data Figs. 2, 4 and 7 are presented with this paper.

Code availability

Custom-written Python scripts that were used to analyse the rotation of mitotic spindles are available from the corresponding author on request.


  1. 1.

    Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

  2. 2.

    Rossi, G., Manfrin, A. & Lutolf, M. P. Progress and potential in organoid research. Nat. Rev. Genet. 19, 671–687 (2018).

  3. 3.

    Artegiani, B. & Clevers, H. Use and application of 3D-organoid technology. Hum. Mol. Genet. 27, R99–R107 (2018).

  4. 4.

    Farin, H. F. et al. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche. Nature 530, 340–343 (2016).

  5. 5.

    Tetteh, P. W. et al. Replacement of lost Lgr5-positive stem cells through plasticity of their enterocyte-lineage daughters. Cell Stem Cell 18, 203–213 (2016).

  6. 6.

    Barriga, F. M. et al. Mex3a marks a slowly dividing subpopulation of Lgr5+ intestinal stem cells. Cell Stem Cell 20, 801–816 (2017).

  7. 7.

    Kon, S. et al. Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes. Nat. Cell Biol. 19, 530–541 (2017).

  8. 8.

    Beumer, J. et al. Enteroendocrine cells switch hormone expression along the crypt-to-villus BMP signalling gradient. Nat. Cell Biol. 20, 909–916 (2018).

  9. 9.

    Serra, D. et al. Self-organization and symmetry breaking in intestinal organoid development. Nature 569, 66–72 (2019).

  10. 10.

    Gehart, H. et al. Identification of enteroendocrine regulators by real-time single-cell differentiation mapping. Cell 176, 1158–1173 (2019).

  11. 11.

    Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).

  12. 12.

    Drost, J. et al. Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. Science 358, 234–238 (2017).

  13. 13.

    Artegiani, B. et al. Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids. Cell Stem Cell 24, 927–943 (2019).

  14. 14.

    Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

  15. 15.

    Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

  16. 16.

    Shimokawa, M. et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545, 187–192 (2017).

  17. 17.

    Cortina, C. et al. A genome editing approach to study cancer stem cells in human tumors. EMBO Mol. Med. 9, 869–879 (2017).

  18. 18.

    Sugimoto, S. et al. Reconstruction of the human colon epithelium in vivo. Cell Stem Cell 22, 171–176 (2018).

  19. 19.

    Essers, J. et al. Analysis of mouse Rad54 expression and its implications for homologous recombination. DNA Repair 1, 779–793 (2002).

  20. 20.

    Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2016).

  21. 21.

    Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).

  22. 22.

    Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

  23. 23.

    Schiroli, G. et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell 24, 551–565 (2019).

  24. 24.

    Betermier, M., Bertrand, P. & Lopez, B. S. Is non-homologous end-joining really an inherently error-prone process? PLoS Genet. 10, e1004086 (2014).

  25. 25.

    Guo, T. et al. Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing. Genome Biol. 19, 170 (2018).

  26. 26.

    Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P. & Del Bene, F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 24, 142–153 (2014).

  27. 27.

    Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

  28. 28.

    Lackner, D. H. et al. A generic strategy for CRISPR–Cas9-mediated gene tagging. Nat. Commun. 6, 10237 (2015).

  29. 29.

    Schmid-Burgk, J. L., Honing, K., Ebert, T. S. & Hornung, V. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat. Commun. 7, 12338 (2016).

  30. 30.

    He, X. et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res. 44, e85 (2016).

  31. 31.

    Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015).

  32. 32.

    Hu, H. et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 175, 1591–1606 (2018).

  33. 33.

    Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458–466 (2013).

  34. 34.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

  35. 35.

    Fujii, M. et al. Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition. Cell Stem Cell 23, 787–793 (2018).

  36. 36.

    Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).

  37. 37.

    Yin, X. et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat. Methods 11, 106–112 (2014).

  38. 38.

    Treyer, A. & Musch, A. Hepatocyte polarity. Compr. Physiol. 3, 243–287 (2013).

  39. 39.

    Wang, T., Yanger, K., Stanger, B. Z., Cassio, D. & Bi, E. Cytokinesis defines a spatial landmark for hepatocyte polarization and apical lumen formation. J. Cell Sci. 127, 2483–2492 (2014).

  40. 40.

    Lazaro-Dieguez, F. & Musch, A. Cell-cell adhesion accounts for the different orientation of columnar and hepatocytic cell divisions. J. Cell Biol. 216, 3847–3859 (2017).

  41. 41.

    Knouse, K. A., Lopez, K. E., Bachofner, M. & Amon, A. Chromosome segregation fidelity in epithelia requires tissue architecture. Cell 175, 200–211 (2018).

  42. 42.

    Wang, M. J., Chen, F., Lau, J. T. Y. & Hu, Y. P. Hepatocyte polyploidization and its association with pathophysiological processes. Cell Death Dis. 8, e2805 (2017).

  43. 43.

    Guidotti, J. E. et al. Liver cell polyploidization: a pivotal role for binuclear hepatocytes. J. Biol. Chem. 278, 19095–19101 (2003).

  44. 44.

    Margall-Ducos, G., Celton-Morizur, S., Couton, D., Bregerie, O. & Desdouets, C. Liver tetraploidization is controlled by a new process of incomplete cytokinesis. J. Cell Sci. 120, 3633–3639 (2007).

  45. 45.

    Aylon, Y. & Oren, M. p53: guardian of ploidy. Mol. Oncol. 5, 315–323 (2011).

  46. 46.

    Vogel, C., Kienitz, A., Hofmann, I., Muller, R. & Bastians, H. Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy. Oncogene 23, 6845–6853 (2004).

  47. 47.

    Duncan, A. W. et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467, 707–710 (2010).

  48. 48.

    Duncan, A. W. et al. Frequent aneuploidy among normal human hepatocytes. Gastroenterology 142, 25–28 (2012).

  49. 49.

    Kurinna, S. et al. p53 regulates a mitotic transcription program and determines ploidy in normal mouse liver. Hepatology 57, 2004–2013 (2013).

  50. 50.

    Duncan, A. W. et al. Aneuploidy as a mechanism for stress-induced liver adaptation. J. Clin. Invest. 122, 3307–3315 (2012).

  51. 51.

    Zhang, J. P. et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 18, 35 (2017).

  52. 52.

    Mitzelfelt, K. A. et al. Efficient precision genome editing in iPSCs via genetic co-targeting with selection. Stem Cell Rep. 8, 491–499 (2017).

  53. 53.

    Zhang, J. Z. et al. A human iPSC double-reporter system enables purification of cardiac lineage subpopulations with distinct function and drug response profiles. Cell Stem Cell 24, 802–811 (2019).

  54. 54.

    Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  55. 55.

    Janda, C. Y. et al. Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Nature 545, 234–237 (2017).

  56. 56.

    Artegiani, B. et al. Generation of knock-in human organoids by CRISPR-HOT. Protoc. Exch. (2020).

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We thank G. Darmasaputra for help with imaging and analyses of polyploid divisions and M. Galli for advice on hepatocyte ploidy; Y. Bar-Ephraïm and J. Bernink for help with FACS; H. Gehart and V. Hornung for providing plasmids; and S. van den Brink for support with preparation of medium components. We acknowledge all of the anonymous tissue donors. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. B.A. was supported by a FEBS long-term fellowship and is the recipient of a VENI grant (NWO-ALW 863.15.015).

Author information




Conceptualization: B.A. and D.H.; methodology: B.A., D.H. and H.C.; software: B.A., D.H., R.K. and X.Z.; formal analysis: B.A., D.H., R.K. and X.Z.; investigation: B.A., D.H., J.B., R.K., I.J. and X.Z.; resources: S.C.v.S.L., S.T., J.v.Z. and H.C.; data curation: B.A., D.H. and R.K.; writing—original draft: B.A., D.H. and H.C.: writing—review and editing: B.A., D.H., J.B., R.K., X.Z. and H.C.; visualization: B.A., D.H. and R.K.; supervision: H.C.; project administration: B.A. and D.H.; and funding acquisition: B.A., S.T., J.v.Z. and H.C. B.A. and D.H. contributed equally as joint first authors. J.B. and and R.K. contributed equally as joint second authors.

Corresponding author

Correspondence to Hans Clevers.

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Competing interests

H.C. holds several patents on organoid technology. Their application numbers, followed by their publication numbers (if applicable), are as follows: PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930; PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990, WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797, WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716, n/a; and GB1819224.5, n/a.

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Extended data

Extended Data Fig. 1 Schematic overview of NHEJ and HDR targeting plasmids used in this study.

List of plasmids used or generated in this study for gene tagging, including a schematic map of the plasmid, the application and the plasmid source.

Extended Data Fig. 2 FACS analysis of knock-in efficiencies mediated by NHEJ or HDR.

a, Gating strategy and representative plot of how transfected and knocked-in cells were defined based on the negative control (non-transfected cells). b, Representative FACS plots of HDR- and NHEJ-mediated knock-in in presence or absence of a dominant negative form of TP53 (DN TP53) when targeting the TUBB locus in human hepatocyte organoids. The experiment was repeated three times independently with similar results. c, Representative images of electroporated human liver ductal organoids for both HDR- and NHEJ-mediated in-frame knock-in of the respective tag at the KRT19 locus, showing knocked-in cells overlapping within the transfected cells. The experiment was repeated at least five times independently with similar results. d, Bar plot showing fold changes of knock-in efficiency between HDR and NHEJ in presence or absence of a dominant negative form of TP53 (DN TP53) in human liver ductal organoids (targeting the KRT19 locus) and human hepatocyte organoids (targeting the TUBB locus. For liver ductal organoids: n=6 for HDR-DN TP53, n=4 for HDR+DN TP53, n=5 for NHEJ-DN TP53, n=3 for NHEJ+DN TP53, for hepatocyte organoids: all conditions n=3 biological independent experiments. Two-sided unpaired t-test performed on the non-normalized data as shown in Fig. 1b-c; *p<0.05; **p<0.01; ***p<0.001. e, Representative brightfield and fluorescent images of HDR- and NHEJ-mediated knocked-in mNEON at the TUBB locus in human hepatocyte organoids. Scale bars, c: 200 µm; e: 20 µm. Numerical source data for d are provided in Statistical Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Representative sequencing results of generated human knock-in organoid lines in this study.

Sequencing results from clonal organoid lines spanning the insertion site for KRT18::mNEON a, CHGA::mNEON b, MUC2::mNEON c, AFP::mNEON d, TUBB::GFP e, CDH1::tdTomato f. For every targeted locus, sequencing results from 1 of the tagged lines is shown.

Extended Data Fig. 4 CDH1-tagged and TUBB-tagged human hepatocyte organoids as a tool for tracing cell movement and mitotic spindle dynamics.

a, Tagging of CDH1 reveals that hepatocytes tend to form rosette structures around lumina (indicated by the white arrows) (left). Representative example of analysis of cell movement in CDH1::mNEON human hepatocyte organoids (right). Individual cell movements were traced based on changes of cell centroid positioning over time. The green dots represent the initial position of the cell centroid and length and orientation of each green arrow represent individual cell centroid movement from begin to end of the experiment. The red line indicates lumen movement. (t = 9 hours, 45 min intervals). The experiment was repeated five times independently with similar results. b, Representative examples of tracing of cell movement based on changes of cell centroid positioning over time in CDH1::mNEON (left) and CDH1::tdTomato (right) human hepatocyte organoids. Initial and final positioning and cell shape outline are represented in blue and pink, respectively, and cell movements are indicated by arrows. Similarly, the initial and final position of the lumen is outlined and its movement is visualized. Note that hepatocytes tend to rotate around the lumen. c, Bar plot showing the average cell rotation for individual organoids with a lumen (light green) or without a lumen (dark green). Note that in organoids in which there is no lumen cells tend to rotate less. as previously mentioned, organoids with a lumen tend to rotate. n=9 clonal organoids with a lumen and n=7 clonal organoids without a lumen. Two-sided unpaired t-test; *p<0.05. d, Example of the determination of mitotic spindle dynamics in TUBB::GFP human hepatocyte organoids. The mitotic spindle orientation was traced over time by marking the initial (gray) and final (red) spindle orientation. The thin line indicates the average position of the spindle poles at every time point between the two. Scale bars, a: left: 75 µm, right: 100 µm; d: 100 µm. Statistics source data are provided in Statistical Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 Characterization of cells with a monopolar spindle in TUBB-tagged human hepatocyte organoids.

a, Representative snapshots of a time-lapse experiments showing the formation and fate of monopolar spindles in TUBB::GFP human hepatocyte organoids, resulting in cell bursting. b, Representative snapshots of a time-lapse experiments in which organoids were incubated with NucRed Dead 647 confirming that cells with a monopolar spindle die. c, Cells with a monopolar spindle stain positive for cleaved caspase-3. All experiments were repeated at least twice independently with similar results. Scale bars, a: 10 µm; b: left: 10 µm, right: 5 µm; c: 5 µm.

Extended Data Fig. 6 Types of non-canonical mitotic spindles in WT and TP53-/- human hepatocyte organoids.

a, Examples of mitotic spindles with disorganized microtubules in a WT (i) and TP53-/- (ii, iii) background. b, Loss of TP53 causes frequent formation of multipolar spindles. c, Example of a non-canonical mitotic event where a spindle pole is lost in a TP53-/- background. All experiments were repeated at least thirteen times independently with similar results. Scale bars, a-c: 5 µm.

Extended Data Fig. 7 Characterization of the impact of TP53 mutation on the phenotype of human hepatocyte organoids.

Representative examples of whole-mount (left) and a zoomed-in focal plane (right) staining of TUBB::GFP TP53-/- organoids showing the intactness of the MRP2-marked bile canalicular network a, and maintenance of hepatocyte polarity as marked by ZO-1, associated with a cell division with a multipolar spindle b, For a-b: experiments were repeated at least twice independently with similar results. c, Box-plot showing the amount of mitosis in WT and TP53-/- TUBB-tagged hepatocytes. Each dot (n) represents mitosis quantification in one single organoid. Mitosis in organoids from 2 donors were quantified. Donor 1, WT: n=5; Donor 2, WT: n=6; Donor 1, TP53-/-: n=10; Donor 2, TP53-/-: n=4 biologically independent experiments. d, Box-plot showing similar TUBB::mNEON fluorescence intensity between WT and TP53-/- TUBB::mNEON hepatocyte organoids. n=3 biologically independent samples per condition. Box-plot elements shown in c-d are minimum, maximum, centre, 25th percentile, 75th percentile. For c-d: Two-sided unpaired t-test. Scale bars, a: left: 10 µm, inset: 5 µm; b: left: 10 µm, right: 5 µm. Statistics source data for c-d are provided in Statistical Source Data Extended Data Fig. 7. Source data

Extended Data Fig. 8 Schematic overview of all targeted loci in this study.

a, Representation of all the genes that have been tagged in this study showing a schematic of the gene pre and post knock-in with all the different tags. Black boxes represent exons, lines represent introns. b, Overview of all the genes that have been tagged in the different organoid cultures in this study.

Supplementary information


Representative examples of time-lapse imaging of TUBB-tagged WT human hepatocyte organoids from two donors.


Representative examples of time-lapse imaging of TUBB::mNEON; CDH1::tdTomato WT human hepatocyte organoids.


Representative examples of time-lapse imaging of TUBB-tagged TP53−/− human hepatocyte organoids from two donors.

Reporting Summary

Supplementary Video 1

Representative examples of time-lapse imaging of TUBB-tagged WT human hepatocyte organoids from two donors.

Supplementary Video 2

Representative examples of time-lapse imaging of TUBB::mNEON; CDH1::tdTomato WT human hepatocyte organoids.

Supplementary Video 3

Representative examples of time-lapse imaging of TUBB-tagged TP53−/− human hepatocyte organoids from two donors.

Source data

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Source Data Fig. 7

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 4

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Source Data Extended Data Fig. 7

Statistical source data.

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Artegiani, B., Hendriks, D., Beumer, J. et al. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing. Nat Cell Biol 22, 321–331 (2020).

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