Abstract
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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Acknowledgements
This study was funded by a major technological innovation project in Hubei province (2017ACA174). Plasmid pRGEB32 was kindly provided by Dr. Qin Hu (Hubei University).
Supporting information
Supplementary Table S1—Primers used for the synthesis of crRNAs in the present study.
Supplementary Table S2—Primers used in the present study.
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Dong, M., Wang, F., Lv, B. et al. CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease. Biotechnol Lett 43, 899–907 (2021). https://doi.org/10.1007/s10529-020-03064-z
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DOI: https://doi.org/10.1007/s10529-020-03064-z