Deaminase-mediated multiplex genome editing in Escherichia coli

Satomi Banno, Keiji Nishida, Takayuki Arazoe, Hitoshi Mitsunobu, Akihiko Kondo

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool 1, 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 lethality 3, 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.

Original languageEnglish
Pages (from-to)423-429
Number of pages7
JournalNature Microbiology
Volume3
Issue number4
DOIs
Publication statusPublished - 1 Apr 2018

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Clustered Regularly Interspaced Short Palindromic Repeats
Cytosine
Guide RNA
Uracil-DNA Glycosidase
Cytidine Deaminase
Escherichia coli
Transposases
Mutation
Thymine
Eukaryota
Mutagenesis
Genes
Nucleotides
Genome
Bacteria
Growth
Gene Editing

Cite this

Banno, Satomi ; Nishida, Keiji ; Arazoe, Takayuki ; Mitsunobu, Hitoshi ; Kondo, Akihiko. / Deaminase-mediated multiplex genome editing in Escherichia coli. In: Nature Microbiology. 2018 ; Vol. 3, No. 4. pp. 423-429.
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Deaminase-mediated multiplex genome editing in Escherichia coli. / Banno, Satomi; Nishida, Keiji; Arazoe, Takayuki; Mitsunobu, Hitoshi; Kondo, Akihiko.

In: Nature Microbiology, Vol. 3, No. 4, 01.04.2018, p. 423-429.

Research output: Contribution to journalArticle

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