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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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).
The authors declare no competing financial interests.
Integrated supplementary information
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.
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.
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.
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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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). https://doi.org/10.1038/nprot.2018.042
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