New pegRNA overcomes transient delivery bottleneck in precise genome editing

Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNAs (pegRNAs), which append a reverse transcription template and primer binding site (RTT–PBS) to the 3’ end of a CRISPR-Cas guide RNA. Although delivery of PE ribonucleoproteins (RNPs) holds substantial therapeutic potential, their limited genome-editing activity constrains clinical applications.

Recently, a team led by Chunqing Song at Westlake University, China, published a study in Nature Biomedical Engineering titled “Boosting prime editing with engineered non-canonical pegRNAs.”

The study developed non-canonical pegRNAs (npegRNAs) that successfully overcome the bottleneck of transient-delivery efficiency in prime editing, markedly enhancing efficiency and stability without increasing off-target risk. These npegRNAs are especially well-suited to RNP and RNA delivery systems required for clinical translation.

Gene-editing technologies, particularly those within the CRISPR–Cas toolkit, herald a new era of precision medicine and biological research by enabling targeted DNA modifications in mammalian cells. Prime editing is a subclass of CRISPR–Cas tools distinguished by its ability to enact accurate DNA changes without inducing double-strand breaks, offering a safer alternative for gene correction.

These tools excel at a range of edits, including base substitutions, insertions, and deletions. The prime editing system operates with a fusion of Streptococcus pyogenes (Sp) Cas9 nickase (Cas9n) and a reverse transcriptase (RT), guided by a pegRNA—an extended single-guide RNA (sgRNA) bearing a 3’ RTT–PBS extension.

Recent efforts have aimed to improve efficiency by advancing core PE proteins—such as hyPE, PE2*, PEmax, PE6, and PE6d—and by incorporating DNA- or RNA-binding motifs, including the RNA-binding motif La fused in PE7. While pegRNAs specify the DNA target and encode the intended edit, their 3’ extensions are vulnerable to cellular exonucleases, compromising the integrity of the RTT–PBS that is critical for PE5.

Figure 1. Design of npegRNAs that improves PE efficiency. (FANG, Guo-Qing, et al., 2026)

Engineered pegRNAs (epegRNAs) provide nuclease resistance via added motifs such as evopreQ1 but inadvertently reduce Cas9 binding. A split pegRNA system, in which the sgRNA and RTT RNA function separately, demonstrates the feasibility of separating editing components, though it is generally less effective than canonical pegRNAs. Because of its transient activity, PE RNP delivery is theoretically effective and safe for in vivo genome editing, yet its practical efficiency remains low and lags behind DNA or mRNA delivery methods across various cell types, hindering the clinical use of PE for treating genetic diseases.

Here, the researchers introduce structurally guided engineering of PE complexes using non-canonical pegRNAs (npegRNAs), integrating the RTT–PBS into sgRNA loops to enhance PE efficiency. This approach yields improved precise editing rates across diverse genomic loci and cell types and enhances therapeutic gene correction in a mouse model of tyrosinemia. Cas9-associated npegRNAs are more resistant to exonuclease degradation, potentially boosting the targeting efficiency of PE complexes in living cells.

With PE RNP delivery, npegRNAs achieved on average a 26.8-fold higher editing yield than standard pegRNAs and a 5.9-fold increase over engineered pegRNAs (epegRNAs). Moreover, npegRNA-mediated RNPs increased the efficiency of installing disease-associated mutations by up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, these findings establish a potent PE strategy and highlight the therapeutic potential of npegRNAs in prime editing applications.