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

Abstract

Gene tagging with fluorescent proteins (FPs) is essential to investigate the dynamic properties of cellular proteins. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology (CRISPR/Cas9) technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest 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 FP, (ii) perturbation of the fusion protein by the FP or (iii) non-specific 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 not only time-and cost-effective to perform whole genome sequencing of each cell clone, but also there are 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 Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics which takes between 6-9 weeks. Using this pipeline, 70% of the targeted genes could be tagged homozygously with FPs and resulted in physiological levels and phenotypically functional expression of the fusion proteins. In . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/188847 doi: bioRxiv preprint first posted online Sep. 14, 2017; 2 contrast to a study that systematically tagged genes using CRISPR/Cas9 in human stem cells 1 , our approach resulted in homozygously tagged proteins of interests. have developed a validation pipeline for endogenously tagged cells which is described in this protocol. INTRODUCTION Paired Cas9D10A nickase approachAll nucleases used for genome editing in mammalian cells (e.g. Zinc Finger Nucleases, Transcription Activator-like Effector Nuclease, CRISPR/Cas9) trigger a dsDNA break at a specific genomic locus which can be repaired by two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology directed repair (HDR). To generate knock-in cell lines, HDR is essential to insert the tag of interest which is done in the presence of an exogenously introduced repair template during the dsDNA break [5][6][7][8][9][10][11][12][13][14] . CRISPR/Cas9 systems consist of either wildtype Cas9 or mutant Cas9 which perform either dsDNA breaks or nicking of single-stranded DNA, respectively, guided by small gRNAs. The paired Cas9D10A nickase approach is the most suitable method for generating endogenously tagged cell lines, as it avoids off-target effects while providing effective...

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins

“…To endogenously tag the condensin I subunit CAP‐D2 and the condensin II subunit CAP‐D3 at the C‐terminus with mEGFP in HeLa Kyoto cells, the paired CRISPR/Cas9 nickase approach was used as described in (Koch et al , ). After selecting GFP‐positive cells by flow cytometry, single‐cell clones were confirmed to be homozygously mEGFP‐tagged at the endogenous locus by PCR, Southern blot and Western blot, respectively.…”

“…Finally, GFP immunoprecipitation and immunoblotting against all subunits of the condensin complex showed that the formation of pentameric condensin complexes was not prevented by the introduced tag in both asynchronous and nocodazole‐arrested mitotic cell populations. The details of all cell line validation steps are described in Koch et al ().…”

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

“…The CRISPR modified cell lines that we used were heterozygotes with a single allele of the gene edited. There is agreement that some genes (estimated by Koch et al, 2018; to be~25%) cannot tolerate a tag at both loci. It has been reported, for example, that lamin B could be modified at both alleles, but actin cannot (Roberts et al, 2017).…”

“…There is agreement that some genes (estimated by Koch et al, 2018; to be~25%) cannot tolerate a tag at both loci. There is agreement that some genes (estimated by Koch et al, 2018; to be~25%) cannot tolerate a tag at both loci.…”

Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors

“…Heterozygous tagging is sufficient to analyze protein localization and dynamics, but not to quantify protein levels or to verify the functionality of the tagged protein. For experiments that require homozygous edits, other approaches can be used (Koch et al., ; Paquet et al., ).…”