Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

DNA double-strand breaks (DSBs) are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes. Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining (NHEJ) system and ultimately cause genome mutagenesis. Here, we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli. The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis . This process introduces chromosome DNA excision with different sizes, which can be manipulated by regulating the activity of host-exonuclease RecBCD. The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein from λ phage. Furthermore, we also found single nucleotide substitutions at the DNA junction, suggesting that T4 DNA ligase, as a single component non-homologous end joining system, has great potential in genome mutagenesis, genome reduction and genome editing.

Background Microbial mutagenesis is an important avenue to acquire microbial strains with desirable traits for industry application. However, mutagens either chemical or physical used often leads narrow library pool due to high lethal rate. The T4 DNA ligase is one of the most widely utilized enzymes in modern molecular biology. Its contribution to repair chromosomal DNA damages, therefore cell survival during mutagenesis will be discussed. Results Expression of T4 DNA ligase in vivo could substantially increase cell survival to ionizing radiation in multiple species. A T4 mediated survival-coupled mutagenesis approach was proposed. When polyhydroxybutyrate (PHB)-producing E . coli with T4 DNA ligase expressed in vivo was subjected to ionizing radiation, mutants with improved PHB production were acquired quickly owing to a large viable mutant library generated. Draft genome sequence analysis showed that the mutants obtained possess not only single nucleotide variation (SNV) but also DNA fragment deletion, indicating that T4 DNA ligase in vivo may contribute to the repair of DNA double strand breaks. Conclusions Expression of T4 DNA ligase in vivo could notably enhance microbial survival to excess chromosomal damages caused by various mutagens. Potential application of T4 DNA ligase in microbial mutagenesis was explored by mutating and screening PHB producing E. coli XLPHB strain. When applied to atmospheric and room temperature plasma (ARTP) microbial mutagenesis, large survival pool was obtained. Mutants available for subsequent screening for desirable features. The use of T4 DNA ligase we were able to quickly improve the PHB production by generating a larger viable mutants pool. This method is a universal strategy can be employed in wide range of bacteria. It indicated that traditional random mutagenesis became more powerful in combine with modern genetic molecular biology and has exciting prospect. Electronic supplementary material The online version of this article (10.1186/s12934-019-1160-7) contains supplementary material, which is available to authorized users.