Abstract
Regenerative therapies require availability of an abundant healthy cell source which can be achieved by efficient 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 efficient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64-modified 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 specific functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9-VP64 pretreated cells ([Formula: see text]) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively affect the post-thaw viability. Co-delivering chemically modified dCas9-VP64 mRNA with gRNAs is an efficient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet effective way to accelerate a wide array of cellular research and translational medical applications.
Published Version
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