Fusing NHP6A to reverse transcriptase improves the efficiency of prime-editing substitutions, insertions, and deletions
Background: Reverse transcriptase shapes prime-editing performance
A study reports that fusing the small protein module NHP6A to reverse transcriptase increased the intended DNA changes produced by prime editing. The researchers reported improvements in substitution, insertion, and deletion editing, as well as compatibility with existing editing tools. These findings come from gene-editing experiments and should be distinguished from evidence of therapeutic benefit in patients.
Prime editing (PE) is a technology for introducing small, precise changes into DNA. Its efficiency can be limited in human cells, so improving its performance remains a challenge. This study focused on reverse transcriptase (RT), a major determinant of PE performance. Drawing on how natural polymerases bind their substrates, the researchers adopted an approach that fuses polymerase substrate-binding proteins (PSBPs) to RT.
Key findings: NHP6A identified in a 12-candidate screen and combined with existing tools
When the researchers tested 12 PSBP candidates, several increased editing efficiency, with the small HMG-box module NHP6A highlighted as a leading candidate. Fusing NHP6A to RT increased intended substitutions, insertions, and deletions. Unwanted insertions and deletions (indels) were reported to remain low. Intended insertion or deletion edits must be distinguished from indels arising as byproducts when interpreting the results.
This improvement could also be used with other PE tools. Broad compatibility was reported with PE3, which adds a single-guide RNA (sgRNA) that directs a nick in one DNA strand; PE4, which uses MLH1dn to transiently suppress mismatch repair; and epegRNA, which has a structured extension at the 3′ end of the prime-editing guide RNA (pegRNA).
Further improvement was also reported when the approach was combined with PE7, which stabilizes the pegRNA tail using La protein. The researchers used NHP6A and La together to introduce four clinically relevant alleles with high editing-product purity. These results concern the experimental introduction of those genetic variants; they do not mean that disease variants were corrected in patients or that symptoms improved. Activity was also reported to be retained when the PSBP fusion was incorporated into a single-component prime editor.
Implications and outlook: Editor design with a small accessory module
The study shows that attaching a small accessory module to RT could be a design option for improving PE performance. Compatibility with existing tools and the additional effect of combining NHP6A with La provide a basis for evaluating multiple improvement strategies together. However, the findings should not be generalized as establishing the extent of improvement for every combination or superiority at all targets.
The available material consists of the bibliographic record and abstract of the paper published in Nucleic Acids Research. The abstract does not provide the cell types used, numerical editing-efficiency and product-purity values for each target, statistical results, or details of the specific alleles. The report of low indel levels alone cannot establish reduced off-target editing or overall safety. The available material also does not establish whether binding free energy or changes in reaction rates were measured directly. Assessing the scope of the approach requires quantitative performance data and detailed experimental conditions.
This account of the research is based on the provided Europe PMC bibliographic record and abstract.
Prime editing (PE) enables precise small DNA changes yet often shows modest efficiency in human cells. Recent advances indicate that the activity of the reverse transcriptase (RT) component is a key determinant of PE performance. Here, we adapt a principle from natural polymerases by fusing a polymerase substrate binding protein (PSBP) with RT to enhance PE. In a twelve-member screen, multiple PSBPs increased editing efficiency, with a compact HMG box module, NHP6A, emerging as a representative lead fusion. NHP6A fused to RT significantly increases intended edits across substitutions, insertions, and deletions while indels remain low. The enhancement is broadly compatible with a nicking sgRNA (PE3), transient mismatch repair inhibition via MLH1dn (PE4), and structured 3' pegRNA extensions (epegRNA). The PSBP module also synergizes with the La protein-assisted pegRNA tail stabilization (the PE7 system) for additional improvements. Using NHP6A together with La, we efficiently installed four clinically relevant alleles with high product purity. Finally, we validate that PSBP fusion can be consolidated into a single component prime editor without loss of activity. These results establish PSBP fusion as a precise route to improve prime editing outcomes and support integrating compact accessory modules into next-generation PE platforms.
When designing prime-editing experiments to introduce a particular variant, researchers could consider the NHP6A–RT fusion as a candidate for comparison with existing editors. Stabilization strategies using La protein and combinations with other PE improvement tools are also candidates for testing. This use case would compare increases in intended editing alongside the formation of unwanted byproducts.
This is a proposal for follow-up research derived from the findings. Introducing four clinically relevant alleles does not by itself establish therapeutic efficacy or suitability for use in patients. Evaluating its value for therapeutic development requires further assessment of reproducibility in relevant cells, quantitative efficiency and product purity at each target, off-target editing, and delivery conditions. Retained activity after integration into a single-component editor also cannot be taken as evidence that delivery or manufacturing challenges have been resolved.