Our workflow for the introduction of a tyrosinemia-related SNP c.786G>A (p.Trp262Ter) into the FAH (fumarylacetoacetate hydrolase) gene began with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provided a homogeneous, undifferentiated starting population. We used electroporation to deliver Cas9-sgRNA together with the homology-directed repair (HDR) template, an ssDNA donor template of 200 nucleotides in length encoding the SNP of interest. 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. Following electroporation, we screened the population of edited hiPS cells using our own SNP detection system. Single cells were seeded and expanded to generate clonal cell lines, and the lines were screened to identify clones with the desired c.786G>A substitution. No preselection was required prior to screening.
- 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
Introducing a tyrosinemia-related SNP 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 pathway in mammalian cells. The editing events could range from insertion of long sequences encoding fusion tags or expression cassettes to single base changes that mimic single-nucleotide polymorphisms (SNPs) related to human diseases. The use of hiPS cells to create isogenic cell lines from either healthy or sick individuals offers the unique potential to study how a specific genetic modification can influence gene function. First, hiPS cells can be generated from a healthy individual and differentiated into the desired cell type. Then, the healthy hiPS cell population can be edited to insert a known or potential disease-relevant mutation and expanded clonally to create an isogenic cell line that only differs from the healthy cells by the introduced mutation. Alternatively, a diseased hiPS cell line can be generated from a sick individual, and gene editing can be used to correct the mutation for the development of therapeutic applications. These edited hiPS cell lines are renewable sources of diseased and healthy cells that are controlled for genetic variability, and they can be used for a variety of downstream applications to study and treat disease. Here, we describe our workflow for the introduction of a disease-related SNP into an endogenous gene.
sgRNA and ssDNA design
A good experimental design is crucial for efficient and successful gene editing. We use sgRNAs with an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. For this project, we selected two sgRNAs with cut sites that were close to the base we wanted to modify (in exon 10 of the FAH gene). We tested both sgRNAs independently. For the HDR template, we used a short oligonucleotide encoding the SNP with 99-nucleotide homology arms related to the insertion site. The Cas9-sgRNA RNP complex and ssDNA donor were introduced to the hiPS cells via electroporation.
Analysis of edited population
Following gene editing, we used our own SNP screening system to determine which of the two sgRNAs generated the higher level of knockin. The fluorescent signal is proportional to editing events at the target site and indicates introduction of the SNP. Analysis of the overall edited population of hiPS cells showed that cells edited with sgRNA 1 had the desired SNP, as indicated by the fluorescent signal above the background.
Characterization of edited clonal cell lines
Since the desired SNP was detected in the pool of cells edited with sgRNA 1, cells from this population were individually seeded using limiting dilution, and then expanded into edited clonal cell lines using our DEF-CS single-cell cloning system. Forty-five days after seeding, clonal cell lines were interrogated for the c.786G>A SNP using our fluorescence-based SNP screening system, which allowed us to rapidly and accurately screen a large number of clones in a 96-well format. Approximately 19% of the clonal cell lines generated a positive fluorescent signal, indicating insertion of the SNP. Nonclonal samples are marked with an asterisk.
We further characterized the positive clonal cell lines via Sanger sequencing and flow cytometry. We used Sanger sequencing to determine if positive clones were homozygous or heterozygous for the SNP. Several clonal cell lines that were homozygous for the SNP were further expanded in our culture system, and their pluripotency was checked by flow cytometry using Oct-4, TRA-1-60, and SSEA-4 as markers. All clonal cell lines exhibited high levels of the three markers, indicating that the clonal hiPS cell lines maintained pluripotency following genome editing.
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