Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study in Dietzia sp. Strain DQ12-45-1b

Abstract

Nonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNA Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNA by NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach in sp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, an strain also showed NHEJ activity when the Ku and LigD of sp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms. Many nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway in sp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency

“…Therefore, errors that are prone to occur in NHEJ are not errors that occur at the outset. In fact, NHEJ is a key strategy for stabilizing the genome, which plays an important role in the repair of DSBs (Lu et al, 2018). Although HDR is more accurate, it has a specific cycle limit.…”

Membrane vesicles from a Dietzia bacterium containing multiple cargoes and their roles in iron delivery

“…We utilized three kinds of media for cultivating Dietzia sp. DQ12‐45‐1b and its mutant strains in this study: (i) GPY medium (glucose, 10 g/l, yeast extract 5 g/l and tryptone 10 g/l) was used to harvest the cell biomass of DQ12‐45‐1b, Δ DtmbtB and Δ DtmbtB :: DtmbtB , (ii) MFN medium, composed of minimal medium [MF medium, Na 2 HPO 4 ·12H 2 O 17.9 g/l, NaH 2 PO 4 ·2H 2 O 7.8 g/l, (NH 4 ) 2 SO 4 5 g/l, KCl 5 g/l, MgSO 4 ·7H 2 O 0.2 g/l, CaCl 2 0.05 g/l, ZnSO 4 ·7H 2 O 0.1 mg/l, MnCl 2 ·4H 2 O 0.03 mg/l, H 3 BO 3 0.3 mg/l, CoCl 2 ·6H 2 O 0.2 mg/l, CuCl 2 ·2H 2 O 0.01 mg/l, NiCl 2 ·6H 2 O 0.02 mg/l, Na 2 MoO 4 ·2H 2 O 0.03 mg/l, pH 8.0] and 5 g/l sodium acetate (NaAc) was used to grow DQ12‐45‐1b amended with different types of MVs (Lu et al ., 2018) and (iii) MFNI medium, MFN medium amended with 8 μM FeCl 3 was used for cultivation of DQ12‐45‐1b and its mutant strains to generate the iron‐limiting condition, and isolation of their MVs. The incubation of DQ12‐45‐1b was all conducted at 30°C, 150 rpm.…”

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

“…Bacterial NHEJ systems have been less extensively studied [14][15][16] . Fortunately, NHEJ systems are present in a diverse group of bacteria, from Mycobacterium tuberculosis to Pseudomonas aeruginosa, and some systems have been used for engineering in their native context 17,18 . Relatively little work has been done to express these systems heterologously, but one study showed NHEJ was able to circularize transformed linear DNA in E. coli 19 and another E. coli study showed NHEJ can repair DSBs caused by Cas9 12 .…”