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.

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Complete ammonia oxidizers (comammox), as novel microbial communities, are predicted to play an important role in the nitrogen cycle. Here we reported the presence of complete nitrification in tidal sediments and examined the diversity and abundance of comammox in natural ecosystems. Metagenome and metatranscriptome of the enrichment culture from tidal sediments harbored the genes of comammox. Near-complete comammox AmoA/B/C- and Hao-like sequences showed close relationships to the known comammox (with sequence identity from 79 to 99%) rather than classical betaproteobacterial ammonia-oxidizing bacteria (β-AOB) (57 to 66%) and ammonia-oxidizing archaea (AOA) (24 to 38%). To analyze the diversity of comammox in natural environments, a new primer set targeting clade A comammox Nitrospira (COM-A) amoA genes was designed based on sequences obtained in this study and sequences from published database. In silico evaluation of the primers showed the high coverage of 89 and 100% in the COM-A amoA database. Application of the primers in six different ecosystems proved their strong availability. Community composition of COM-A suggested a relatively higher diversity than β-AOB in similar environments. Quantification results showed that COM-A amoA genes accounted for about 0.4-5.6% in total amoA genes. These results provide novel insight into our perception of the enigmatic comammox and have significant implications for profound understanding of complex nitrification process.

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