Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA; University of California San Diego, La Jolla, California, USA.
Halıcıoğlu Data Science Institute, University of California San Diego, La Jolla, California, USA; University of California San Diego, La Jolla, California, USA.
CRISPR J. 2022 Apr;5(2):294-310. doi: 10.1089/crispr.2021.0131. Epub 2022 Mar 28.
Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.
腺嘌呤碱基编辑器 (ABEs) 已经经历了多轮诱变，旨在优化其作为高效、精确的基因组编辑工具的功能。尽管 ABE 突变体及其相应的 DNA 或 RNA 编辑活性的数据集不断扩大，但定义这些变化的分子机制仍有待阐明。在这项研究中，我们使用基于熵的分类模型，该模型依赖于现有蛋白质序列的进化数据，对这些突变的性质进行了系统的解释。我们使用该模型结合实验分析，鉴定了两个先前报道的突变，它们在 ABE 的 RNA 编辑功能景观中形成了一个上位性对。分子动力学模拟揭示了这两个突变如何通过单独和协同作用影响底物结合和催化活性的原子细节，从而深入了解这两个突变如何通过上位性相互关联的机制。
