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

Li, L., Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China
Uygun, B., Massachusetts General Hospital and Harvard Medical School
Yarmush, M. L., MGH/Harvard Medical School
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.