A Multiplex Genome Editing Method for Escherichia coli Based on CRISPR-Cas12a

Various methods for editing specific sites in the chromosome are available, and gene-size (∼1 kb) integration into a single site or to introduce deletions, short insertions or point mutations into multiple sites can be conducted in a short period of time. However, a method for rapidly integrating mu...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Frontiers in microbiology 2018-10, Vol.9, p.2307-2307
Hauptverfasser: Ao, Xiang, Yao, Yi, Li, Tian, Yang, Ting-Ting, Dong, Xu, Zheng, Ze-Tong, Chen, Guo-Qiang, Wu, Qiong, Guo, Yingying
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:Various methods for editing specific sites in the chromosome are available, and gene-size (∼1 kb) integration into a single site or to introduce deletions, short insertions or point mutations into multiple sites can be conducted in a short period of time. However, a method for rapidly integrating multiple gene-size sequences into different sites has not been developed yet. Here, we describe a method and plasmid system that makes it possible to simultaneously insert genes into multiple specific loci of the genome without the need for chromosomal markers. The method uses a CRISPR-Cas12a system to eliminate unmodified cells by double-stranded DNA cleavage in conjunction with the phage-derived λ-Red recombinases to facilitate recombination between the chromosome and the donor DNA. We achieved the insertion of up to 3 heterologous genes in one round of recombination and selection. To demonstrate the practical application of this gene-insertion method, we constructed a recombinant producing an industrially useful chemical, 5-aminolevulinic acid (ALA), with high-yield. Moreover, a similar two-plasmid system was built to edit the genome of the extremophile .
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2018.02307