Effective Genome Editing with ISDra2 TnpB and Deep Learning-Predicted omegaRNAs

Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified so far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on- and off-target editing and to devise a deep learning model, termed TEEP (TnpB Editing Efficiency Predictor), capable of predicting ISDra2 TnpB guiding RNA (omegaRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3 % in the murine liver and 65.9 % in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the advancements and tools presented in this study facilitate the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics.