UGT1A9 plays a role in the regulation of cellular homeostasis by limiting drug-induced stress. Our workflow for adding a myc tag to the UGT1A9 gene began with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provided a homogeneous, undifferentiated starting population. 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. Together with the RNP complex, we electroporated a 200-nucleotide long ssDNA encoding the myc tag. After editing, cells from the now heterogeneous population (consisting of wildtype, knockout, and successfully tagged cells) were single-cell isolated in a 96-well plate and expanded to generate clonal cell lines. The clonal cell lines were then screened to identify clones with the correct insertion.
- 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 a myc tag in hiPS cells
One of the most powerful applications of genome editing is the introduction of precise changes in 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 insert an epitope tag to detect proteins for which no good antibody is available, or to identify protein interaction networks using affinity fusion-based protein purification. Here, we describe the workflow we developed for introducing an epitope tag to an endogenous gene.
sgRNA and ssDNA design
For successful homologous recombination, the cut site of the sgRNA should be as close as possible to the targeted insertion site: the end of exon 5 of UGT1A9. For this project, there were three sgRNAs that could be used, and we tested them in independent reactions. We used our Guide-it sgRNA In Vitro Transcription Kit to synthesize sgRNAs that had an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. For the HDR template, we ordered an ssDNA from an oligonucleotide synthesis company that encoded the myc tag with 99-nucleotide homology arms related to the UGT1A9 insertion site. The Cas9-sgRNA RNP complex (prepared by co-incubating Guide-it Recombinant Cas9 protein with in vitro-transcribed sgRNA) and HDR template were introduced to the hiPS cells via electroporation.
Analysis of edited population
Following genome editing, we used our Guide-it Knockin Screening Kit to detect successful, full-length HDR events in the pool of edited cells and to determine which of the three sgRNAs generated the highest level of knockin. With our system, two different fluorescent signals correlate to correct insertion of the myc tag in the target site; green fluorescence indicates correct insertion at the 5' end, and red fluorescence indicates correct insertion at the 3' end. The highest signal was obtained from the population electroporated with the HDR template and an RNP complex containing sgRNA 2; therefore, this population was chosen for subsequent single cell isolation.
Characterization of clonal cell lines
Cells edited with sgRNA 2 were individually sorted using flow cytometry, and then expanded into edited clonal cell lines using our DEF-CS single-cell cloning system. Sixteen days after seeding, clonal cell lines were interrogated for myc-tag insertion using our knockin screening kit. Out of 230 clones, three (clones 62, 129, and 168) were positive for a correct insertion at both ends (green and red fluorescence could be detected). Three other clones (191, 193, and 220) were only positive for a correct 5' insertion (only green fluorescence could be detected), suggesting insertion of a truncated myc tag.
These results were confirmed using the Guide-it Indel Identification Kit followed by Sanger sequencing. All six clones were heterozygous, with one allele encoding the wild type sequence. Clonal cell lines 62, 129, and 168 also had one allele with correct and full insertion of the myc tag. Therefore, despite a low knockin efficiency, we were able to correctly identify edited clones with our knockin screening system.
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