Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany.
Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
Nucleic Acids Res. 2024 Jul 22;52(13):8017-8031. doi: 10.1093/nar/gkae481.
Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing Cre discrimination between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the plasticity of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations were used to investigate the possible mechanisms behind the specificity switch. Our findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.
Cre/loxP 重组系统的转化研究集中在通过修饰 Cre/DNA 相互作用来提高其特异性。尽管进行了广泛的努力，但 Cre 区分底物的确切机制仍然难以捉摸。Cre 识别 13 个 bp 的反向重复序列，在 8 bp 的间隔区启动重组。虽然文献表明，当两个 lox 位点都具有匹配的间隔子时，lox 位点之间具有非-loxP 间隔序列的有效重组会发生，但缺乏对此假设的实验验证。为了填补这一空白，我们研究了loxP 8 bp 间隔区相同对的靶位点变化，筛选了 6000 个独特的loxP 样序列。这些位点中约有 84%表现出有效的重组，证实了间隔序列对催化的可塑性。然而，某些间隔子会对重组产生负面影响，强调了序列的依赖性。对重组效率低下的间隔子进行 Cre 的定向进化不仅产生了具有增强活性的重组酶，还产生了具有重新编程的选择性活性的突变体。鉴定了改变间隔特异性的突变，并且使用分子建模和动力学模拟来研究特异性开关背后的可能机制。我们的研究结果突出了为间隔序列特异性精细调整位点特异性重组酶的潜力，为用于基因组工程应用的设计重组酶的应用特性提供了一个新的概念。
