Background

A study published in Nature Biotechnology reports improved gene-editing efficiency by optimizing the terminal structure of prime editing guide RNA (pegRNA). The researchers evaluated 2,858 RNA motifs in human cells and selected candidates that outperformed a widely used motif in correcting multiple variants. The study systematically compared and improved guide RNA structure, an additional focus for optimization beyond editing proteins.

Prime editing (PE) systems have improved through engineering of both proteins and small RNA components. Structured RNA motifs appended to the 3′ end of pegRNA are considered important determinants of guide stability and editing efficiency. However, according to the paper’s abstract, these motifs have not been studied sufficiently. The study asked which terminal motifs yield better editing performance and how they can be optimized.

Key findings

The researchers developed PE-PRISM, a high-throughput pooled screen to discover and optimize 3′ RNA motifs for pegRNA in human cells. A total of 2,858 motifs were evaluated across four iterative libraries. Candidates included naturally derived and engineered pseudoknots, G-quadruplexes, and reverse transcriptase recruitment elements.

After the initial screen, the team refined candidates using RNA structure-informed mutagenesis and combinatorial variant screening. This process yielded the engineered and evolutionarily optimized pseudoknot variants tevo2.0, eHAV, and eSBRMV1-A. The approach went beyond adding stabilizing motifs by repeatedly assessing candidate structures and variants.

The main comparison came from a screen correcting 847 pathogenic variants listed in ClinVar. The high-performing motifs improved PE efficiency over the widely used tevopreQ1 motif in more than 90% of the edits evaluated. Here, “more than 90%” refers to the proportion of edits for which efficiency improved. It does not mean that correction rates for individual targets exceeded 90%, or that editing efficiency increased by at least 90%. The provided abstract does not report absolute correction rates for individual targets or the average size of the improvement.

The researchers reported higher correction efficiency for disease-associated variants in primary human cells. They also reported improved in vivo editing efficiency in mouse brain and liver. Motifs identified in the cell screen were evaluated in other experimental systems, but these results do not establish therapeutic efficacy in patients.

Implications and outlook

The study establishes pegRNA terminal motifs as a concrete focus for comparison and optimization in prime-editing design. By comparing performance with an existing motif across multiple pathogenic variants and extending evaluation to primary human cells and mouse tissues, it provides a basis for further design studies.

The available material, however, is limited to bibliographic records and the abstract. Changes in motif-specific half-life, effect sizes at individual targets, delivery conditions, and long-term safety are difficult to assess from these sources alone. The evidence also does not support claims that off-target risk has fallen to baseline or that dosing can be reduced. Further validation is needed to establish performance across prime-editing targets and cell conditions before extending the approach to other nucleic acid therapies.

The study methods and main results are described in the paper abstract indexed in Europe PMC.