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Deaminase-mediated multiplex genome editing in Escherichia coli

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 inhibitor6 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.

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Acknowledgements

We would like to thank A. Miyabe and M. Kakimoto for their technical assistance. This work was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical Research and Development (AMED). This work was also partly supported by a Special Coordination Fund for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan; the Cross-ministerial Strategic Innovation Promotion Program; JSPS KAKENHI (grant number 26119710, 16K14654); and the New Energy and Industrial Technology Development Organization (NEDO).

Author information

S.B. and K.N. performed all of the experiments with help from all authors. S.B., K.N. and H.M. wrote the manuscript with input from all authors. T.A. provided technical advice. K.N. and A.K. conceived the project.

Competing interests

A provisional patent has been submitted in part entailing the reported approach.

Correspondence to Keiji Nishida or Akihiko Kondo.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7, Supplementary Tables 1–7.

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DOI

https://doi.org/10.1038/s41564-017-0102-6

Further reading

Fig. 1: Single mutagenesis using Target-AID in bacteria.
Fig. 2: Gain-of-function mutagenesis of the rpoB gene.
Fig. 3: Mutated positions and frequency using different lengths of sgRNAs.
Fig. 4: Multiplex mutagenesis.