瑞士苏黎世大学Gerald Schwank团队近期取得重要工作进展，他们研究提出，利用增强型ISDra2-TnpB系统和深度学习预测ωRNA进行有效基因组编辑。相关研究成果2024年9月23日在线发表于《自然—方法学》杂志上。

据介绍，转座子（IS200/IS605）编码的TnpB蛋白是2类V型CRISPR效应子的前身，已成为迄今为止鉴定出的最紧凑的基因组编辑器之一。在

研究人员优化了耐辐射奇球菌（ISDra2）TnpB在哺乳动物细胞（TnpBmax）中的应用设计，使编辑效果平均提高了4.4倍。此外，研究人员开发了在K76位突变的变体，可以识别替代靶邻近基序（TAM），扩大了ISDra2-TnpB的靶向范围。研究人员进一步生成了10211个靶位点TnpBmax编辑效率的广泛数据集。

这使得能够描绘出目标内和目标外编辑的规则，并设计出一个深度学习模型，称为TnpB编辑效率预测器（TEEP；https://www.tnpb.app)，能够高效预测ISDra2-TnpB引导RNA（ωRNA）的活性（r>0.8）。使用TEEP，在腺相关病毒（AAV）载体递送TnpBmax后，研究人员在小鼠肝脏和小鼠大脑中实现了高达75.3%和65.9%的编辑效率。

总之，这一研究提出的一套工具促进了TnpB作为超紧凑可编程内切酶在研究和治疗中的应用。

附：英文原文

Title: Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs

Author: Marquart, Kim Fabiano, Mathis, Nicolas, Mollaysa, Amina, Mller, Saphira, Kissling, Lucas, Rothgangl, Tanja, Schmidheini, Lukas, Kulcsr, Pter Istvn, Allam, Ahmed, Kaufmann, Masako M., Matsushita, Mai, Haenggi, Tatjana, Cathomen, Toni, Kopf, Manfred, Krauthammer, Michael, Schwank, Gerald

Issue&Volume: 2024-09-23

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.

DOI: 10.1038/s41592-024-02418-z

Source: https://www.nature.com/articles/s41592-024-02418-z