Engineering high-efficiency mutants of the transposase ISDra2 TnpB via protein-generative models and energy-optimized design

Transposon-associated TnpB is a compact and versatile gene editor that holds significant promise in the life sciences. However, current engineering strategies for TnpB are limited, resulting in low cleavage efficiency in mammalian cells that restricts its broader use. In this study, we developed a method termed TnpB-GMRE, which integrates a generative protein model for TnpB with a virtual screening pipeline based on the minimum recovery rate and energy minimization. From 100,000 generated sequences, we selected the top five candidates for experimental assessment. Without the use of enrichment strategies such as flow cytometry or antibiotic selection, three of the five mutants displayed higher editing activity across four target sites. Among them, the TnpB-TD mutant achieved the highest editing activity (17.7%), representing a 50% increase compared to the original ISDra2 TnpB from Deinococcus radiodurans . In addition, the TnpB-TD mutant exhibited greater diversity in editing profiles and superior efficiency in deleting long fragments (> 10 bp) relative to the intact ISDra2 TnpB. Molecular dynamics simulations revealed that compared to the wild type, the TnpB-TD mutant adopted a greater number of low-energy conformational states and displayed an increased positive charge on its surface, suggestive of a stabilized structure that may underlie its enhanced editing performance. This strategy provides a framework for optimizing compact nucleases and broadens the available toolkit for gene-editing applications.