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.
- Pluripotent stem cells
Gene editing in hiPS cells
- Tagging an endogenous gene with AcGFP1 in hiPS cells
- Tagging an endogenous gene with a myc tag in hiPS cells
- Generating clonal hiPS cell lines deficient in CD81
- Introducing a tyrosinemia-related SNP in hiPS cells
- Inserting an expression cassette into the AAVS1 locus in hiPS cells
- Editing hiPS cells using electroporation
- Editing hiPS cells using gesicle technology
- Single-cell cloning of hiPS cells
- Beta cells
- Neural stem cells
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Tagging an endogenous gene with AcGFP1 in hiPS cells
One of the most powerful applications of genome editing is the introduction of precise changes at specific sites, which exploits the homology-directed repair (HDR) pathway in mammalian cells. The editing event could range from a single base change to the insertion of longer sequences like fusion tags or expression cassettes. Endogenous gene tagging can be used to generate reporter cell lines, which are important for studying and understanding cellular activities. Tagging genes to create fluorescent fusion proteins enables monitoring of protein subcellular localization as well as protein dynamics and regulation in live cells. Such edited cells can be extremely useful models for studying protein function, as well as for drug screening or drug discovery studies. Here, we describe our workflow for tagging an endogenous gene with a fluorescent protein in hiPS cells.
sgRNA and ssDNA design
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). The sgRNA has an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. For the HDR template, we synthesized 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 and HDR template encoding AcGFP1 were introduced to hiPS cells via electroporation.
Analysis of edited population
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.
Characterization of clonal cell lines
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.
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