For CRISPR applications (with only DNA plasmids): Day 1: Seed 1.5×10^5 cells per well on a 6 well cell culture dish in 2 ml OptimemI medium to starve the cells prior transfection. Incubate the cells at 37ºC, 5% CO2 for one day prior to the transfection. The cells should be at least 50% confluency during the transfection. Day 2: Ensure the cells are at least 60% confluent during transfection. Dilute the chosen gRNA plasmids and donor plasmid in 200 µl of jetPrime buffer, with: - CRISPR/Cas9 nickase sense: 0.5 µg - CRISPR/Cas9 nickase antisense: 0.5 µg - Donor plasmid: 1 µg - Buffer: 200 µl - JetPrime: 4 µl. Mix well and add 4 µl of jetPrime solution, incubate at RT for 15min and add transfection complex onto the cells. After 4h at 37ºC and 5% CO2 incubation replace transfection medium with normal growth medium and grow cells until they are ~80% confluent.

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.