RNA-Based dCas9–VP64 System Improves the Viability of Cryopreserved Mammalian Cells

Hu Yong, Li Lei, Yu Yin, Huang Haishui, Uygun Basak E., Yarmush Martin L.

Regenerative therapies require availability of an abundant healthy cell source which can be achieved by e±cient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly e±cient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64- modi¯ed mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell speci¯c functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9- VP64 pretreated cells (P < 0:05) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively a®ect the post-thaw viability. Co-delivering chemically modi¯ed dCas9-VP64 mRNA with gRNAs is an e±cient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet e®ective way to accelerate a wide array of cellular research and translational medical applications.


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January, 2018


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  • CRISPR/Cas9 Antibody
    CRISPR/Cas9 Antibody


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    Clermont-Ferrand, France
    Jul 10-Jul 12, 2024


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