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.

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.

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.

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.

We developed a complete workflow for tagging endogenous genes with epitope tags. Our workflow starts with footprint-free CRISPR/Cas9-mediated editing to tag the gene of interest, followed by single-cell cloning of the edited population and rapid screening of expanded clonal cell lines to identify positive clones. Importantly, we can use our knockin detection system to quickly identify the correct clones in a high-throughput manner, even when the knockin efficiency is low.