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 tool. 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. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) 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 in combination with a degradation tag (LVA tag) 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.

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

“…To evaluate the base editing efficiency using Cas9 variants in C. glutamicum , AID was fused to the C‐terminal of the nickase form of VQR‐Cas9 and VRER‐Cas9 using a 100 aa linker, generating nVQR‐Cas9(D10A)‐AID and nVRER‐Cas9(D10A)‐AID, respectively. Although UGI that inhibits removal of uracil (an immediate product of cytidine deamination) from DNA is commonly used in eukaryotic base editing, its expression in bacteria may increase genome‐wide nonspecific mutagenesis and compromise genome integrity (Banno et al, ). Since we previously demonstrated that high‐efficiency base editing can be achieved without UGI in C. glutamicum (Y. Wang et al, ), UGI was not used for constructing base editors in this study.…”

Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids

“…55 Further validation of some BTK parts, such as the dCas9 and Cas9 systems, is needed to conclude that they will function reliably across diverse species. Other established broad-host-range tools—such as Tn7-transposon integration 46 , Group II intron-based gene disruption 57 , and emerging CRISPR methods for targeted mutagenesis 58 —could also be incorporated into the BTK-compatible Golden Gate framework in the future.…”

Impact of CRISPR interference on strain development in biotechnology

“…Recently, CRISPR‐mediated nucleotide editing has been developed and as discussed below this method may also be used for manipulating gene expression strength. The first method described was called deaminase‐mediated targeted nucleotide editing (Target‐AID) [170] and was applied in the bacteria E. coli [171,172] and C. glutamicum [173]. Target‐AID relies on a cytidine deaminase fused to dCas9 and introduces C:G to T:A mutations at sequences defined by the sgRNA [170].…”

“…Target‐AID relies on a cytidine deaminase fused to dCas9 and introduces C:G to T:A mutations at sequences defined by the sgRNA [170]. Target‐AID enables a high mutation efficiency and even simultaneous multiplex editing without introducing lethal double‐stranded DNA breaks, requiring a foreign DNA as a template and is independent of inefficient homologous recombination [171,173]. One application is the introduction of premature translational stop codons resulting in loss‐of‐function mutations [173].…”