(191aa) Chemically Modified mRNA Based CRISPR-Cas9 System Improves the Viability of Cryopreserved Mammalian Cells

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 a 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 significant 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 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.