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Nat Microbiol. 2018 Apr;3(4):423-429. doi: 10.1038/s41564-017-0102-6. Epub 2018 Feb 5.

Deaminase-mediated multiplex genome editing in Escherichia coli.

Author information

1
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, Japan.
2
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, Japan. keiji_nishida@people.kobe-u.ac.jp.
3
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
4
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, Japan. akondo@kobe-u.ac.jp.
5
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Hyogo, Japan. akondo@kobe-u.ac.jp.

Abstract

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool1, 2. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality3, 4. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) 5 adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor 6 in combination with a degradation tag (LVA tag) 7 resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.

PMID:
29403014
DOI:
10.1038/s41564-017-0102-6
[Indexed for MEDLINE]

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