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

Abstract

Gene tagging with fluorescent proteins is essential to investigate 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 permits 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 either (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) non-specific genomic DNA damage by CRISPR/Cas9. In this protocol1, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.\n\nWe have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair with minimal off-target effects. It is time- and cost-consuming to perform whole genome sequencing of each cell clone. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics. This protocol takes between 6-9 weeks. Using this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins and result in physiological levels and phenotypically functional expression of the fusion proteins.\n\nEditorial SummaryThis protocol provides a detailed workflow describing how to insert fluorescent markers into all alleles of a gene of interest using CRISPR/Cas 9 technology and how to generate and validate homozygous fluorescent knock-in cell lines.