The team led by Yin Hao from Wuhan University in China published a research paper titled "Rapid generation of long, chemically modified pegRNAs for prime editing" in the journal Nature Biotechnology. The study used RNA ligation technology to successfully break through the length barrier of chemically synthesized RNA and efficiently prepared high-purity chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). The L-epegRNA prepared by this method showed excellent editing efficiency in multiple cell lines and two primary cells.
The research team first used sgRNA as a model and RNA ligation method to verify the feasibility and efficiency of T4 Rnl2 enzyme in RNA ligation. In order to further improve the ligation efficiency, the research team conducted a series of optimization experiments and comprehensively optimized the RNA ligation system from multiple dimensions such as ligation temperature, splint DNA length, ligation ratio and enzyme amount. These optimization measures improved the RNA ligation efficiency. And the RNA generated by this method successfully induced gene editing.
Figure 1. Overview of the initial design scheme for ligation of pegRNA. (Lei X, et al., 2024)
In order to more effectively protect the 3' end of pegRNA, the research team designed three different connection strategies, and finally found that when the evopreQ1 motif is used in combination with chemical modification, it can show better performance and achieve the highest prime editing efficiency. In addition, in order to remove the by-products present in the connection, the research team also introduced high-performance liquid chromatography (HPLC) technology, which can accurately and efficiently separate and purify the target pegRNA from the reaction mixture to a higher purity. HPLC-purified L-epegRNA further improved the editing efficiency. It is worth noting that even L-epegRNA that has not been purified by HPLC has shown satisfactory editing efficiency. This means that L-epegRNA that induces efficient prime editing can be prepared without HPLC purification experimental conditions.
The research team also systematically optimized the ribonucleoprotein (RNP) delivery system and compared the performance of the optimized RNP and plasmid delivery in various cells. The results showed that in most comparative experiments, L-epegRNA-mediated RNP delivery showed higher editing efficiency than plasmid delivery.
As another strategy widely used in the field of treatment and scientific research, RNA delivery has also attracted much attention. Using electroporation transfection technology, the research team successfully achieved co-delivery of PEmax mRNA and L-epegRNA, and achieved efficient editing in various cell lines. At the same time, the research team also tested L-epegRNA in two primary cells and demonstrated excellent editing ability. Compared with in vitro transcribed epegRNA, L-epegRNA increased the efficiency of prime editing by up to hundreds of times. This indicates that it has great application potential in clinical applications, precision medicine and genetic disease treatment.
Figure 2. PE efficiencies using L-epegRNA in human primary T cells and hematopoietic stem cells. (Lei X, et al., 2024)
Finally, the research team also explored the ability of this method to generate longer RNA. L-epegRNA of 210 nucleotides and 234 nucleotides were successfully prepared, and efficient long fragment insertion was achieved using RNP and RNA delivery systems, respectively.
Reference
Lei X, et al. Rapid generation of long, chemically modified pegRNAs for prime editing. Nature Biotechnology, 2024: 1-12.