Our workflow for knocking out the CD81 gene begins with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provides a homogeneous, undifferentiated starting population. To edit the hiPS cells, 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. Since CD81 is a protein in the extracellular membrane, FACS sorting can be used to select CD81-knockout cells from the edited population, and then single cells can be seeded and expanded to generate clonal cell lines.

A good experimental design is crucial for efficient and successful gene editing. In order to knock out the CD81 gene, we chose an sgRNA targeting the first exon. The sgRNA was in vitro transcribed and had an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. After incubating the sgRNA with Cas9 protein, the Cas9-sgRNA RNP complex was introduced to hiPS cells via electroporation.

A week after the gene editing experiment, cells were analyzed using flow cytometry. Since CD81 is a membrane protein, staining the cells with an anti-CD81 antibody labeled with FITC gave a direct and accurate percentage of the cells deficient in CD81, which is equivalent to successful gene editing by CRISPR/Cas9. Almost 90% of the cells had CD81 knocked out.

The next phase of the establishment of the edited clonal cell lines was to sort the subpopulation of cells knocked out for CD81 and single-cell isolate them by flow cytometry. Single cells were expanded to clonal cell lines using our DEF-CS single-cell cloning system that ensures a single-cell survival rate higher than 60%. Several clonal cell lines were assessed for knockout of CD81 as well as pluripotency via flow cytometry. All clones exhibited a loss of CD81 expression. Pluripotency was maintained in all of the edited clonal lines, as evidenced by the expression of the three pluripotency markers at levels comparable to those in the parental line.

CRISPR/Cas9-mediated editing and single-cell cloning can be harsh on hiPS cells, and it can force selective pressures that favor unintended mutations that affect the karyotype. To confirm karyotype stability, we examined several of the expanded clonal lines that were deficient in CD81. All lines were found to have normal, stable karyotypes. These data show that our genome editing and single-cell cloning protocols effectively edit and expand hiPS cell clones without introducing karyotypic abnormalities.

Simultaneously, we characterized the specific insertions or deletions (indels) created during the CRISPR/Cas9 editing process in the clonal cell lines using our protocol for indel identification, followed by Sanger sequencing. Because Cas9-induced double-strand breaks are mainly repaired via the error-prone NHEJ repair pathway, clonal cell lines can exhibit different indels in each allele, and these indels can vary between cell lines. The results showed that each clone had a unique set of indels that led to the knockout of CD81.

We have developed a complete workflow for the knockout of an endogenous gene. Our workflow consists of footprint-free CRISPR/Cas9-mediated editing, single-cell cloning of the edited population, and characterization of expanded clonal cell lines to identify positive clones. The edited hiPS cells maintain pluripotency and have normal, stable karyotypes, which are important for downstream applications such as directed differentiation.

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