Protocol | Published:

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

Nature Protocols volume 13, pages 14651487 (2018) | Download Citation

Abstract

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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Acknowledgements

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

Author notes

    • Birgit Koch
    •  & Yin Cai

    Present addresses: Max Planck Institute for Medical Research, Heidelberg, Germany (B.K.); Roche SIS, Waiblingen, Germany (Y.C.).

Affiliations

  1. EMBL, Heidelberg, Germany.

    • Birgit Koch
    • , Bianca Nijmeijer
    • , Moritz Kueblbeck
    • , Yin Cai
    • , Nike Walther
    •  & Jan Ellenberg

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jan Ellenberg.

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DOI

https://doi.org/10.1038/nprot.2018.042

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