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Updated: Jul 29, 2026

Protocols for C-Brick DNA Standard Assembly Using Cpf1
Published on: June 15, 2017
CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease
Mengjie Dong1, Fei Wang1, Bin Lv1
1State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, School of Life Sciences, Hubei University, Wuhan, People's Republic of China.
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.
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