Exploring CRISPR-Mediated Activation of Lineage-Specific Genes for Direct Cellular Reprogramming Using RNPs

DNA- and Virus-free activation of pluripotency networks offers potential for regenerative medicine and cellular engineering. In this study, a ribonucleoprotein (dRNP)-based CRISPR activation (CRISPRa) system was used to transiently upregulate endogenous transcription factors and non-coding RNAs in human adult and neonatal fibroblasts. Fibroblasts were transfected with dRNP complexes targeting the canonical Yamanaka factors (OCT4, SOX2, KLF4, MYC, LIN28), additional regulators (REX1, NANOG), and the long non-coding RNA MIR302CHG. Optimization included variations in factor combinations, guide RNA design, transfection frequency, small molecules, and medium conditions. Quantitative PCR confirmed robust transcriptional induction of pluripotency factors and pri-miR-302/367, demonstrating engagement of both coding and non-coding networks. Morphological observations suggested early cellular responses to pluripotency activation under optimized conditions. These results indicate that dRNP-mediated CRISPRa can reliably induce transient activation of multiple endogenous pluripotency genes and non-coding RNAs in human fibroblasts, providing a non-integrative, transient, and potentially safer method for probing early cellular responses relevant to reprogramming and cellular engineering.