Cas9의 절반 크기로 비슷한 기능을 하는 단백질 TnpB가 세포핵으로 잘 이동하도록 구조를 최적화해 유전자편집 효율을 최대 4.4배 높인 연구결과가 공개됐다.
더불어 TnpB 기반 유전자가위 작동을 예측하는 AI 모델을 개발해 생쥐 간에서 최대 75.3%, 생쥐 뇌에서 65.9%의 유전자 교정 효율을 달성했다.
Abstract
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 thus 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-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app ), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) 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 set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics.
Nat Methods. 2024.09.23.
