Our workflow for tagging tubulin with AcGFP1 began with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provides a homogeneous, undifferentiated starting population. We used electroporation to deliver Cas9-sgRNA together with the HDR template, an ssDNA donor template encoding AcGFP1. We delivered the Cas9-sgRNA complex in the form of ribonucleoprotein (RNP) in order to decrease off-target effects and for footprint-free genome editing. We used our own system to synthesize a long ssDNA donor template that has a reduced tendency to randomly integrate and a low cytotoxic response after being delivered to cells, as compared to dsDNA. Since tubulin is endogenously expressed in hiPS cells, edited cells could be isolated using flow cytometry (below). AcGFP1+ cells were single-cell isolated by flow cytometry, seeded, and expanded to generate clonal cell lines.

A good experimental design is crucial for efficient and successful gene editing. In order to tag tubulin at its N-terminus with AcGFP1, we chose an sgRNA targeting exon 2 (below). We used our Guide-it sgRNA In Vitro Transcription Kit to produce an sgRNA has an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. For the HDR template, we used the Guide-it Long ssDNA Production System to synthesize a long ssDNA encoding AcGFP1 with 350-nucleotide-long homology arms. We synthesized both sense and antisense ssDNA donor templates and tested them independently. The Cas9-sgRNA RNP complex (produced by co-incubating Guide-it Recombinant Cas9 and in vitro-transcribed sgRNA) and HDR template encoding AcGFP1 were introduced to hiPS cells via electroporation.

Following genome editing, the hiPS cells were analyzed via flow cytometry to determine knockin efficiency, which could be directly correlated to the percentage of AcGFP1+ cells. FACS analysis showed 2.8% or 1.4% AcGFP1+ cells, depending on the HDR template used (antisense or sense, respectively), indicating efficient knockin of AcGFP1 into the N-terminus of tubulin.

AcGFP1+ hiPS cells were individually sorted into 96-well plates using flow cytometry, and then expanded into edited clonal cell lines using our DEF-CS single-cell cloning system. Clonal lines were characterized by flow cytometry and fluorescence microscopy. For each clonal cell line, >99% of cells were AcGFP1+. When observed under the microscope, the localization of AcGFP1 correlated with the known expression pattern of tubulin, suggesting correct fusion of AcGFP1 to tubulin.

We have developed a complete workflow for tagging endogenous genes to generate reporter cell lines. Our workflow starts with footprint-free CRISPR/Cas9-mediated editing to tag the gene of interest, followed by successful screening and single-cell cloning of edited hiPS cells to generate clonal cell lines that express an AcGFP1-tubulin fusion protein. To view more details and complete data for this Case Study, read our tech note.