Deaminase-mediated multiplex genome editing in Escherichia coli

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

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.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cytidine Deaminase / genetics
  • Cytidine Deaminase / metabolism*
  • DNA Glycosylases / antagonists & inhibitors
  • DNA-Directed RNA Polymerases / genetics
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics
  • Galactokinase / genetics
  • Gene Editing / methods*
  • Genetic Engineering / methods*
  • Genome, Bacterial / genetics
  • Point Mutation / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Transposases / genetics

Substances

  • Escherichia coli Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • rpoB protein, E coli
  • Galactokinase
  • Transposases
  • DNA-Directed RNA Polymerases
  • DNA Glycosylases
  • Cytidine Deaminase