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.

中文翻译：

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Cre/loxP 系统的工程间隔区特异性


Cre/loxP 重组系统的转化研究重点是通过修改 Cre/DNA 相互作用来增强其特异性。尽管付出了巨大的努力，但控制 Cre 基质之间区分的确切机制仍然难以捉摸。 Cre 识别 13 bp 反向重复序列，在 8 bp 间隔区中启动重组。虽然文献表明，当两个 lox 位点都具有匹配的间隔区时，具有非 loxP 间隔区序列的 lox 位点之间会进行有效的重组，但缺乏对这一假设的实验验证。为了填补这一空白，我们研究了相同对的 loxP 8 bp 间隔区的靶位点变异，筛选了 6000 个独特的 loxP 样序列。这些位点中大约 84% 表现出有效的重组，证实了间隔序列对于催化的可塑性。然而，某些间隔区会对重组产生负面影响，强调序列依赖性。 Cre 在低效重组间隔区上的定向进化不仅产生了活性增强的重组酶，而且产生了具有重编程选择性活性的突变体。确定了改变间隔区特异性的突变，并使用分子建模和动力学模拟来研究特异性转换背后的可能机制。我们的研究结果强调了微调位点特异性重组酶以实现间隔序列特异性的潜力，为增强设计重组酶在基因组工程应用中的应用特性提供了一个新颖的概念。