Hefei National Laboratory for Physical Sciences at the Microscale and Department of Physics, University of Science and Technology of China, Hefei, 230026, Anhui, China.
Center for Theoretical Biological Physics, Rice University, Houston, TX, 77005, USA.
Nat Commun. 2021 Nov 11;12(1):6529. doi: 10.1038/s41467-021-26789-5.
Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 - 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the "TC" motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.
碱基编辑器 (BEs) 在基因治疗的医学应用中具有巨大的潜力。然而，高精度的碱基编辑需要 BEs 能够在狭窄的活性窗口（4-10 个核苷酸）内区分靶碱基和多个旁观者碱基。在这里，为了帮助设计这些优化的编辑器，我们提出了一种离散状态随机方法来构建一个分析模型，该模型明确评估编辑靶碱基和旁观者碱基的概率。我们的模型结合全原子分子动力学模拟，再现了 A3A-BE3 及其变体针对“TC”基序和旁观者编辑的实验数据。通过分析这种方法，我们提出了几个可以指导减少旁观者效应的 BE 设计的一般原则。然后将这些原则应用于设计 A3G-BE 中 T218 位置的一系列点突变，以进一步减少其旁观者编辑。我们通过实验验证了新的突变在不同基因组位置减少旁观者编辑的程度提供了不同的严格性，这与我们的理论模型一致。因此，我们的研究提供了一个计算辅助平台，以协助基于科学的减少旁观者效应的 BE 设计。
