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.