Our workflow for knocking in AcGFP1 under the control of the EF1α promoter into the AAVS1 locus 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. 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, as compared to dsDNA. From the overall edited population, AcGFP1+ cells were single-cell isolated by limiting dilution and expanded to generate clonal cell lines.

A good experimental design is crucial for efficient and successful gene editing. We chose an sgRNA targeting the AAVS1 locus. The sgRNA, produced with the Guide-it sgRNA In Vitro Transcription Kit, has an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. For the HDR template, we used our Guide-it Long ssDNA Production System to synthesize a long ssDNA containing the EF1α-AcGFP1 expression cassette with either 300- or 600-nucleotide homology arms related to the AAVS1 site. The Cas9-sgRNA RNP complex (prepared by co-incubating Guide-it Recombinant Cas9 with in vitro-transcribed sgRNA) and HDR template were introduced to hiPS cells via electroporation.

Following gene editing, the cells were analyzed via flow cytometry to determine the percentage of AcGFP1+ cells. FACS analysis showed that 3–4% of the edited population were AcGFP1+, regardless of the length of the homology arms, indicating efficient knockin of the EF1α-AcGFP1 expression cassette.

AcGFP1+ cells were individually seeded using limiting dilution and expanded into edited clonal cell lines using our DEF-CS single-cell cloning system. The expanded clonal lines were characterized by their fluorescence and the correct insertion of the EF1α-AcGFP1 construct in the AAVS1 site. Insertion was confirmed using two pairs of primers; one of the primers in each pair annealed outside the region spanned by the homology arm, in the AAVS1 locus, to avoid false detection of residual repair template. Successful knockin of the EF1α-AcGFP1 expression cassette into the AAVS1 site was detected in 10/11 clonal cell lines. Sanger sequencing of the junctions in the 10 edited cell lines showed a seamless insertion of the long ssDNA donor template; no mutations could be detected and there was full sequence alignment.

We have developed a complete workflow for the targeted knockin of a fluorescent protein expression cassette. Our workflow starts with footprint-free CRISPR/Cas9-mediated editing, followed by single-cell cloning and screening of edited hiPS cells. We have demonstrated that we can generate multiple clonal cell lines that have precise and seamless insertion of the expression cassette in the AAVS1 safe-harbor locus.