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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.


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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).

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Authors and Affiliations



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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