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...
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Veröffentlicht in: | Frontiers in microbiology 2018-10, Vol.9, p.2307-2307 |
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Sprache: | eng |
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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
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ISSN: | 1664-302X 1664-302X |
DOI: | 10.3389/fmicb.2018.02307 |