OBJECTIVE To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. RESULTS We developed a method named as FnCas12a and T5 exonuclease (CT5) cloning system. FnCas12a performs site-directed cleavage to the target DNA fragments, and T5 exonuclease generates 20-30 nt single-stranded region at each end of the DNA fragments for homologous recombination-mediated DNA assembly. CT5 was applied to multi-fragment assembly and DNA cloning of large vectors (> 10 kb). The efficiencies were approximately 91.4% and 97%, respectively. In addition, CT5 cloning is also utilized for the "walking" of DNA elements, which enables subtle modification of the relative distances of DNA elements in plasmids. CONCLUSIONS The CT5 method was a precise and exquisite DNA operating system and provided an ideal platform for the study of gene functions, genetic engineering and synthetic biology.

中文翻译：

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CT5，一种基于 FnCas12a 和 T5 核酸外切酶组合的微妙体外 DNA 组装方法

目的开发一种基于FnCas12a和T5核酸外切酶的DNA组装新方法。结果 我们开发了一种名为 FnCas12a 和 T5 核酸外切酶 (CT5) 克隆系统的方法。FnCas12a 对目标 DNA 片段进行定点切割，T5 核酸外切酶在 DNA 片段的每一端生成 20-30 nt 的单链区域，用于同源重组介导的 DNA 组装。CT5应用于大载体（> 10 kb）的多片段组装和DNA克隆。效率分别约为 91.4% 和 97%。此外，CT5 克隆还被用于 DNA 元件的“行走”，它可以对质粒中 DNA 元件的相对距离进行细微的修改。结论 CT5 方法是一个精确、精致的 DNA 操作系统，为基因功能的研究提供了理想的平台，