Deaminase-mediated multiplex genome editing in Escherichia coli

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. Nuclease-mediated genome editing in bacteria has been limited by toxicity problems linked to DNA cleavage. The use of cytidine deaminase fused to a nuclease-deficient Cas9 bypasses some of these problems, enabling modification of multiple loci simultaneously.