CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease

Objective To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. Results We developed a method named as Fn C as12a 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...

Full description

Saved in:
Bibliographic Details
Published in:Biotechnology letters 2021-04, Vol.43 (4), p.899-907
Main Authors: Dong, Mengjie, Wang, Fei, Lv, Bin, Mei, Meng, Ma, Lixin, Hu, Yunhong, Zhai, Chao
Format: Article
Language:English
Subjects:
Applied Microbiology
Assembling
Assembly
Biochemistry
Biomedical and Life Sciences
Biotechnology
Cloning
Cloning vectors
Deoxyribonucleic acid
DNA
Exonuclease
Fragments
Genetic engineering
Homologous recombination
Homology
In vitro methods and tests
Life Sciences
Microbiology
Original Research Paper
Plasmids
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Objective To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. Results We developed a method named as Fn C as12a 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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-020-03064-z