Optimization of CRISPR/Cas9 technology to knock-out genes of interest in aneuploid cell lines

Dyikanov, D.T.; Vasiluev, P.A.; Rysenkova, K.D.; Aleksandrushkina, N.A.; Tyurin-Kuzmin, P.A.; Kulebyakin, K.Y.; Rubtsov, Y.P.; Shmakova, A.A.; Evseeva, M.N.; Balatskiy, A.V.; Semina, E.V.; Rostovtseva, A.I.; Makarevich, P.I.; Karagyaur, M.N.

Авторы: Dyikanov D.T., Vasiluev P.A., Rysenkova K.D., Aleksandrushkina N.A., Tyurin-Kuzmin P.A., Kulebyakin K.Y., Rubtsov Y.P., Shmakova A.A., Evseeva M.N., Balatskiy A.V., Semina E.V., Rostovtseva A.I., Makarevich P.I., Karagyaur M.N.
Журнал: Tissue Engineering - Part C: Methods
Том: 25
Номер: 3
Год издания: 2019
Издательство: Mary Ann Liebert Inc.
Местоположение издательства: United States
Первая страница: 168
Последняя страница: 175
DOI: 10.1089/ten.TEC.2018.0365
Аннотация: 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 via 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 multi-round 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 alleles disruption and decrease expression of corresponding proteins in cell populations in vitro. However, certain off-target activity was observed as well. Unexpectedly, we didn’t detect the increment of de novo off-target DNA sites cleavage after CRISPR/Cas9 re-use, that proves our approach is suitable for genome editing in aneuploidy cell lines. Our protocol can be used for in vitro models creation via genome editing of aneuploid cells or cells with restricted clonogenic potential.
Добавил в систему: Карагяур Максим Николаевич

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Optimization of CRISPR/Cas9 technology to knock-out genes of interest in aneuploid cell linesстатья

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