Generation and validation of homozygous fluorescent knock-in cells using CRISPR–Cas9 genome editing


Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR–Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) nonspecific genomic DNA damage by CRISPR–Cas9. In this protocol, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.

We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair (HDR) with minimal off-target effects. It is time-consuming and costly to perform whole-genome sequencing of each cell clone to check for spontaneous genetic variations occurring in mammalian cell lines. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern blotting analysis, Sanger sequencing, microscopy, western blotting analysis and live-cell imaging for cell-cycle dynamics. This protocol takes between 6 and 9 weeks. With this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins, thus resulting in physiological levels and phenotypically functional expression of the fusion proteins.

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Figure 1: Paired CRISPR–Cas9 nickase approach.
Figure 2: Validation pipeline of genome-edited cell lines.
Figure 3: FACS of genome-edited cell lines in which NUP358 is tagged at the N terminus.
Figure 4: Junction PCR of HeLa Kyoto mEGFP–NUP358+ cells.
Figure 5: Sanger sequencing at the target site (start codon).
Figure 6: Southern blotting analysis of HeLa Kyoto mEGFP–NUP358+ cell clones.
Figure 7: Western blotting analysis of HeLa Kyoto mEGFP–NUP358+ cells.
Figure 8: Workflow of the analysis of mitotic timing.
Figure 9: Mitotic duration in HeLa Kyoto mEGFP–NUP358+ cell clones.
Figure 10: Live-cell imaging of HeLa Kyoto mEGFP–NUP358+ cell clones.


  1. 1

    Mahen, R. et al. Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells. Mol. Biol. Cell 25, 3610–3618 (2014).

    PubMed  PubMed Central  Google Scholar 

  2. 2

    Otsuka, S. et al. Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope. eLife 5, e19071 (2016).

    PubMed  PubMed Central  Google Scholar 

  3. 3

    Wachsmuth, M. et al. High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells. Nat. Biotechnol. 33, 384–389 (2015).

    CAS  PubMed  Google Scholar 

  4. 4

    Politi, A.Z. et al. Quantitative mapping of endogenously fluorescently tagged proteins using FCS-calibrated four-dimensional imaging. Nat. Protoc. (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Trevino, A.E. et al. Genome editing using Cas9 nickases. Methods Enzymol. 546, 161–174 (2014).

    CAS  PubMed  Google Scholar 

  7. 7

    Pattanayak, V. et al. Determining the specificities of TALENs, Cas9, and other genome-editing enzymes. Methods Enzymol. 546, 47–78 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Dambournet, D. et al. Tagging endogenous loci for live-cell fluorescence imaging and molecule counting using ZFNs, TALENs, and Cas9. Methods Enzymol. 546, 139–160 (2014).

    CAS  PubMed  Google Scholar 

  9. 9

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Doudna, J.A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 346, 1258096 (2014).

    PubMed  Google Scholar 

  12. 12

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Wang, H. et al. CRISPR/Cas9 in genome editing and beyond. Annu. Rev. Biochem. 85, 227–264 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Sternberg, S.H. & Redding, S. DNA interrogation by the CRISPR. Nature 507, 62–67 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat. Commun. 8, 13905 (2016).

    Google Scholar 

  19. 19

    Miyaoka, Y. et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci. Rep. 6, 23549 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Mao, Z. et al. DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells. Neoplasia 11, 683–691 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Bertolini, L.R. et al. Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells. Mol. Biotechnol. 41, 106–114 (2009).

    CAS  PubMed  Google Scholar 

  22. 22

    Chu, V.T. et al. Increasing the efficiency of homology-directed repair for CrIsPr-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Srivastava, M. et al. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell 151, 1474–1487 (2012).

    CAS  Google Scholar 

  24. 24

    Boettcher, R. et al. Efficient chromosomal gene modification with CRISPR/Cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res. 42, e89 (2014).

    CAS  Google Scholar 

  25. 25

    Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. FEBS J. 282, 4289–4294 (2015).

    Google Scholar 

  26. 26

    Greco, G.E. et al. SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV. DNA Repair (Amst) 43, 18–23 (2016).

    CAS  Google Scholar 

  27. 27

    Heijink, A.M. et al. The DNA damage response during mitosis. Mutat. Res. 750, 45–55 (2013).

    CAS  PubMed  Google Scholar 

  28. 28

    Lin, S. et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    PubMed  PubMed Central  Google Scholar 

  29. 29

    Branzei, D. & Foiani, M. Regulation of DNA repair throughout the cell cycle Nat. Rev. Mol. Cell Biol. 9, 297–308 (2008).

  30. 30

    Mao, Z. et al. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 7, 2902–2906 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Pepperkok, R. & Ellenberg, J. High-throughput fluorescence microscopy for systems biology. Nat. Rev. Mol. Cell Biol. 7, 690–696 (2006).

    CAS  PubMed  Google Scholar 

  32. 32

    Landry, J.J.M. et al. The genomic and transcriptomic landscape of a HeLa cell line. G3 (Bethesda) 3, 1213–1224 (2013).

    Google Scholar 

  33. 33

    Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Kim, S. et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    CAS  PubMed  Google Scholar 

  36. 36

    Roberts, B. et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol. Biol. Cell 28, 2854–2874 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Paix, A. et al. Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acids Res. 44, e128 (2016).

    PubMed  PubMed Central  Google Scholar 

  38. 38

    Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

    CAS  PubMed  Google Scholar 

  39. 39

    Lemp, N.A. et al. Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function. Nucleic Acids Res. 40, 7280–7290 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Lukinaviius, G. et al. SiR–Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6, 8497 (2015).

    Google Scholar 

  41. 41

    Heller, C. Principles of DNA separation with capillary electrophoresis. Electrophoresis 22, 629–643 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Sanger, F., Nicklen, S. & Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467 (1977).

    CAS  PubMed  Google Scholar 

  43. 43

    Brinkman, E.K. et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. 44

    Hsu, P.D. et al. DNA targeting specificity of rNA-guided Cas9 nucleases. Nat. Biotechnol. 2, 827–832 (2013).

    Google Scholar 

  45. 45

    Doench, J.G. et al. Optimized sgrNA design to maximize activity and minimize off-target effects of CRISPR-cas9. Nat. Biotechnol. 34, 184–191 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Chen, X. et al. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).

    CAS  PubMed  Google Scholar 

  47. 47

    Held, M. et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nat. Methods 7, 747–754 (2010).

    CAS  PubMed  Google Scholar 

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We thank the mechanical and electronics workshops of EMBL for custom hardware, the Advanced Light Microscopy Facility of EMBL for microscopy support and the Flow Cytometry Core Facility of EMBL for cell sorting. We gratefully acknowledge G. Reid for critical reading of the manuscript. This work was supported by grants to J.E. from the European Commission EU-FP7-Systems Microscopy NoE (grant agreement 258068), EU-FP7-MitoSys (grant agreement 241548) and iNEXT (grant agreement 653706), as well as by the European Molecular Biology Laboratory (B.K., B.N., M.K., Y.C., N.W. and J.E.). N.W. and Y.C. were supported by the EMBL International PhD Programme (EIPP).

Author information




B.K. designed and performed the experiments. B.K. developed the protocol with the help of B.N., Y.C. and N.W. B.N. and M.K. tested the protocol. Y.C. and N.W. created the automation for the cell-cycle analysis. B.K. wrote the protocol with help from B.N., Y.C., N.W. and J.E. All authors contributed to the interpretation of the data and read and approved the final manuscript.

Corresponding author

Correspondence to Jan Ellenberg.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Donor plasmid.

The donor plasmid consists of the fluorescent marker gene (mEGFP in this case) which is flanked by 500-800 bp homology arms of the GOI. A linker is placed between the tag and the gene to maintain functionality of the tagged protein. As a backbone vector pUC-based plasmids are used.

Supplementary Figure 2 Scheme of expected junction PCR result.

A forward primer binding at the 5′ end outside of the left homology arm and a reverse primer binding to the fluorescent marker gene will result in one PCR product of the expected size. To test if all alleles are tagged with the fluorescent marker at the correct locus, a forward primer located 5′ outside of the left homology arm and a reverse primer 3′outside of the right homology arm were used. Two PCR products using this primer set will indicate heterozygote clones and one PCR product which runs at the expected size of the tagged gene will indicate homozygosity.

Supplementary Figure 3 Southern blot transfer of DNA.

This scheme depicts how to build up the southern blot transfer as described in step 48| Option B, step xii-xxiii). Two long filter papers are dipped into a tank filled with 10X SSC and used as wicks. Gel, nylon membrane and filter papers were sandwiched on a glass plate as depicted to transfer the DNA from the gel onto the membrane.

Supplementary Figure 4 Example sequences containing point mutations.

Sanger sequencing was performed with a PCR fragment (lower sequence line #1-278) which was aligned to the expected sequence of the GOI (middle sequence line labeled with #3201-3500). The overlaying green and red lines indicate a point mutation at position 3411 of the expected sequence, i.e. one allele has the nucleotide T at this position (green line) whereas another allele has the nucleotide A (red line) at the same position. This indicates a point mutation (red A) within one of the alleles.

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Supplementary Figures 1–4 (PDF 609 kb)

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Koch, B., Nijmeijer, B., Kueblbeck, M. et al. Generation and validation of homozygous fluorescent knock-in cells using CRISPR–Cas9 genome editing. Nat Protoc 13, 1465–1487 (2018).

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