| dc.description.abstract | Targeted genomic manipulation by the Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease system has the ability to make precise changes in mammalian genomes. Furthermore, the development of mRNA-based delivery has showed great potential in gene editing therapy due to its transient expression without mutant risk, and currently, several clinical trials based on mRNA genetic editing are in progress.
In current thesis, the aim was to produce a more stable and less immunologically triggering Cas9 mRNA to be delivered to human peripheral blood mononuclear cells (PBMCs). We aimed for enhanced precise repair, known as homology directed repair (HDR), by delivering the single-stranded oligodeoxynucleotide (ssODN) repair template 24 hours post first electroporation with single-guide RNA (sgRNA) and Cas9wt mRNA. Cas9wt was also coupled to the chromatin modulating fusion HMGB1 to promote HDR outcomes. Gene editing was detected by droplet digital PCR (ddPCR), and western blot was performed to verify Cas9 protein expression. mRNA was synthesized in vitro and various approaches were performed to obtain precise editing outcomes in desired locus, such as adding 8M of urea to the mRNA synthesis, extended purification of RNA, and adding 40 units of RNase inhibitors prior to electroporation.
Our results demonstrated that time-controlled two-step transfection of Cas9wt mRNA lead to a higher non-homologous end joining (NHEJ) compared to Cas9-HMGB1 mRNA, ∼ 10% versus < 1%, while for HDR, the editing results were close to 0% for Cas9wt mRNA and Cas9-HMGB1 mRNA. Ribonucleoproteins (RNPs) were used as a standard, with an editing outcome between ∼ 20 − 60%. Optimizing the in vitro transcription protocol by adding urea to the mRNA synthesis did not improve precise gene editing (< 1% for HDR and < 3% for NHEJ). The extended purification of RNA did not affect the purity significantly, leading to a decrease in obtained mRNA concentration instead. Finally, testing two different RNase inhibitors resulted in < 5% HDR and < 5% NHEJ.
We conclude that optimization of the in vitro transfection protocol is needed to obtain a more stable mRNA. The inclusion of RNase inhibitors increased the editing of HDR and NHEJ to some extent compared to the other tested conditions, however, the ratio between mRNA and inhibitor added should be further tested out. Preventing the degradation of mRNA remains a great challenge, and tackling this is crucial to provide a foundation for the development of mRNA-based CRISPR therapeutics into a safe, stable, and efficient editing platform. | |
