npegRNA with editing sequences in an internal guide-RNA loop improves prime-editing efficiency
Background: Efficiency limits of RNP delivery
A noncanonical pegRNA (npegRNA), which places editing sequences in an internal loop of the guide RNA, increased editing yield in prime-editing (PE) experiments using ribonucleoprotein (RNP) delivery. The researchers reported higher precise-editing rates across multiple genomic sites and cell types.
PE is a gene-editing technology that uses a prime-editing guide RNA (pegRNA) to make precise genetic changes. In conventional pegRNAs, the reverse-transcription template (RTT) and primer-binding site (PBS) are attached to the 3′ end of the guide RNA.
Delivering PE complexes as RNPs has potential therapeutic applications, but low genomic editing efficiency remains an obstacle. This study examined whether changing guide-RNA structure could improve editing performance with this delivery approach.
Key findings: Internal-loop design and improved editing yield
The researchers developed an npegRNA that incorporates the RTT and PBS into an internal loop of a single guide RNA. This design changes the placement of the sequences used for editing.
When bound to Cas9 protein, npegRNA showed greater resistance to exonuclease degradation. The researchers suggested that this property may have increased the targeting efficiency of PE complexes in living cells. However, the provided abstract does not establish how much longer the complexes remained inside cells.
In PE RNP-delivery experiments, npegRNA editing yield was on average 26.8-fold higher than with conventional pegRNA and 5.9-fold higher than with an engineered pegRNA (epegRNA). These are relative gains under the experimental conditions and do not represent absolute editing rates.
Efficiency also increased in experiments introducing disease-associated variants. In human-cell experiments that included Jurkat T cells and induced pluripotent stem cells (iPSCs), the efficiency of variant introduction with npegRNA-based RNPs was up to 123-fold higher. The abstract does not specify the cell and target combination that produced this maximum. Because these results involve introducing disease-associated variants, they should be distinguished from therapeutic correction that restores disease-associated variants to the normal sequence.
Significance and outlook: From correction in mice to further validation
The researchers also reported improved therapeutic gene correction in a mouse model of tyrosinemia. The change in guide-RNA structure thus improved correction performance in an animal model as well as in cell experiments.
It is too early, however, to interpret these findings as demonstrating therapeutic efficacy for hard-to-treat genetic diseases or establishing a standard for next-generation gene therapy. The available material is the journal article’s abstract; absolute correction rates in mice, symptom improvement, survival, and safety data were outside the scope of the available evidence. Further assessment of absolute editing rates by target, delivery conditions, off-target editing, and toxicity will be needed to determine whether the relative yield gains translate into sufficient performance for practical use.
The study can be consulted in the abstract and bibliographic record of the paper published in Nature Biomedical Engineering.
EMOTION_TAG: interesting
Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.
In studies that edit cells with PE RNPs, npegRNA can be evaluated alongside conventional pegRNA and epegRNA as a candidate design. For example, in experiments creating research models by introducing disease-associated variants into human cells, comparing absolute editing rates at the same target and under the same delivery conditions could help assess the design’s practical value. The improved correction reported in tyrosinemia mice also provides a basis for extending the work to therapeutic research. However, greater efficiency in introducing variants and therapeutic benefit are separate outcomes. Assessing actual therapeutic applications will require determining whether adequate correction rates can be achieved, evaluating off-target editing and toxicity, and checking whether the effects persist.