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 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. We then used our efficient hepatocyte differentiation protocol to generate functional hepatocytes.

A good experimental design is crucial for efficient and successful gene editing. We used our Guide-it sgRNA In Vitro Transcription Kit to synthesize sgRNAs with an optimized scaffold sequence to enhance binding to Cas9 and to 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 (prepared by co-incubating Guide-it Recombinant Cas9 and in vitro-transcribed sgRNA) and ssDNA donor were introduced to the hiPS cells via electroporation.

Following gene editing, we used our Guide-it SNP Screening Kit to determine which of the two sgRNAs generated a higher level of knockin. The fluorescent signal is proportional to editing events at the target site and indicates the 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.

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 the Guide-it Indel Identification Kit followed by 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.

Since tyrosinemia is a liver disorder, we wanted to differentiate the edited cells into a suitable cell type for studying the disorder. Using the Cellartis iPS Cell to Hepatocyte Differentiation System, we differentiated clonal cell line #181 into hepatocytes. On Day 22 after the start of differentiation, both control and edited hiPSC-derived hepatocytes displayed a typical hepatocyte morphology. Immunostaining on Day 29 for the hepatic marker HNFα showed that, on average, >92% of the cells from each line were HNFα-positive, indicating a high differentiation efficiency.

Drug metabolism is a central hepatocyte function. A critical metric for hepatocyte functionality is the expression and activity of drug metabolizing enzymes in the cytochrome P450 (CYP) family. On Day 29 after the start of differentiation, we measured the activities of key CYP enzymes by LC/MS. Both control and edited cell lines showed CYP1A, CYP3A, CYP2C9, CYP2B6, and CYP2D6 activities, which are responsible for 80–90% of clinical drug metabolism.

We have developed a complete workflow for knocking in disease-related SNPs. Our workflow starts with footprint-free CRISPR/Cas9-mediated editing, followed by SNP screening of the edited pool of cells, single-cell cloning of the edited population, and rapid screening of expanded clonal cell lines to identify positive clones. We have demonstrated that we can generate multiple clonal cell lines that have the desired tyrosinemia-related SNP, maintain pluripotency, and retain a normal karyotype. Using our hepatocyte differentiation protocol, we further showed that an edited cell line could be differentiated into hepatocytes to provide a relevant model for studying tyrosinemia.