Flores-Fernández Carol N, Lin Da, Robins Katherine, O'Callaghan Chris A
Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom.
Nucleic Acids Res. 2025 Jun 20;53(12). doi: 10.1093/nar/gkaf548.
Type IIS restriction enzyme-mediated DNA assembly is efficient but has sequence constraints and can result in unwanted sequence scars. To overcome these drawbacks, we developed UniClo, a type IIS restriction enzyme-mediated method for universal and flexible DNA assembly. This is achieved through a combination of vector engineering, DNA methylation using recombinant methylases, and steric blockade using CRISPR-dCas9 technology to regulate this methylation. Type IIS restriction enzyme sites within fragments to be assembled are methylated using recombinant methylases, while the fragment-flanking outer sites are protected from methylation by a recombinant dCas9-sgRNA complex. During the subsequent assembly reaction, only the protected flanking sites are cut as only they are unmethylated. Fragments are correctly assembled, despite containing internal sites for the single type IIS restriction enzyme used for the one-pot assembly. The assembled plasmid can be used as a donor plasmid in a subsequent assembly round with the same type IIS restriction enzyme and the assembly vector engineering ensures removal of potential scars by a trimming process. This simplifies assembly design and only three vectors are required for any multi-round assembly. These vectors all use the same pair of overhangs. UniClo provides a simple scarless approach for hierarchical assembly of any sequence and has wide potential application.
IIS型限制酶介导的DNA组装效率高，但存在序列限制，并且可能会产生不需要的序列疤痕。为了克服这些缺点，我们开发了UniClo，这是一种用于通用和灵活DNA组装的IIS型限制酶介导的方法。这是通过载体工程、使用重组甲基化酶进行DNA甲基化以及使用CRISPR-dCas9技术进行空间位阻以调节这种甲基化的组合来实现的。待组装片段内的IIS型限制酶位点使用重组甲基化酶进行甲基化，而片段侧翼的外部位点则通过重组dCas9-sgRNA复合物保护不被甲基化。在随后的组装反应中，只有受保护的侧翼位点被切割，因为只有它们未被甲基化。尽管片段包含用于一锅法组装的单一IIS型限制酶的内部位点，但片段仍能正确组装。组装好的质粒可以在后续的组装轮次中用作供体质粒，使用相同的IIS型限制酶，并且组装载体工程通过修剪过程确保去除潜在的疤痕。这简化了组装设计，任何多轮组装只需要三种载体。这些载体都使用相同的一对突出端。UniClo为任何序列的分层组装提供了一种简单的无疤痕方法，具有广泛的潜在应用。
