[An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea]

With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.