Tissue Engineering Part C: Methods Vol. 25
Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge
Most commonly used cell lines are readily susceptible to genome editing and present a good object for cell models to establish disease-causing genes and find ways to cure diseases. However, karyotype and phenotype heterogeneity between individual cells in such cultures as well as multiplicity of target alleles make generation of desired cell lines by single-cell cloning (used for diploid cells) inapplicable. We designed and tested a simple approach for targeted genome modification of single cells in sizable cell populations, containing multiple karyotype and phenotype variants. To obtain the cell lines with suppressed expression of target proteins, we applied an original multiround genome modification protocol, monitoring protein expression level and impairment of target and off-target (undesired) DNA cleavage sites. We found that repeated modifications increase efficacy of target DNA allele disruption and decrease expression of corresponding proteins in cell populations in vitro. However, certain off-target activity was observed as well. Unexpectedly, we did not detect the increment of de novo off-target DNA site cleavage after CRISPR/Cas9 reuse, which proves our approach is suitable for genome editing in aneuploidy cell lines. Our protocol can be used for in vitro model creation by genome editing of aneuploid cells or cells with restricted clonogenic potential.