In the post-genomics era [1], essential topics in the bacterial field are exploring gene functions and further strengthening microbial cell factories[2], which depended on manipulating genes through deletion, suppression, or over-expression. So, gene-editing tools and gene interference tools can deeply advance the development of relevant research. Gene-editing tools, as “cutting” approaches, may lead to cytotoxicity due to the formation of DNA double-strand breaks, especially in bacteria that have weak self-repair ability. While gene interference tools, which do not cut targets, can be used in a relatively safe manner to regulate expression in bacteria. Many robust approaches, such as RNAi[3], [4], [5], [6] and CRISPR interference (CRISPRi)[7], [8], [9], [10], [11], have been successfully applied to microbes, but we still believe that different tools with new features, such as being more compact, more specific or without PAM limitation, are still needed to be explored.

Recently, we reported an HpSGN system[12], which is an effective and specific editing tool without sequence limitation for both DNA (Fig. 1, left) and RNA substrates. A notable trait is that the cleavage would be invalid when the base at the position of 1 on the target mismatches to the hpDNA (the guiding DNA probe in HpSGN). We inferred that this characteristic can be taken to build the hpDNA-assisted structure-guided nuclease mediating interference (HpSGNi) system. The HpSGNi was composed of a FEN1 nuclease (~35 kDa) and mis-hpDNAs (~45-55 nt), which contains three mutations on the guide sequence at the position of 1, 2, and 3 (Fig. 1, right). The FEN1 captured the mis-hpDNAs due to their stem-loop structures. Although the cleavage is invalid, FEN1 can still be guided by mis-hpDNA to locate on target loci.

This HpSGNi in mammalian cells has been successfully validated when targeting RNA substrate [13], but not studied when targeting DNA substrate in bacterial hosts. So as designed (Fig. 1, right), when the mis-hpDNAs are located on the promoter region or coding region, they could guide the FEN1 to form a FEN1-hpDNA-target ternary complex, which prevented the binding and moving forward of RNAP. The transcription was then hindered and mediated the silencing of the target gene’s expression in microbes. The feasibility of the constructed HpSGNi system will be tested in vitro and in Escherichia coli (E. coli) in this study.

For this HpSGNi, the capture/binding has no requirement like PAM or PFS for the targets’ sequence. The compact size of the FEN1-coding plasmid is potentially to be beneficial for delivery. Our findings based on studies in E. coli may be beneficial for the downregulation of gene expression in other bacterial hosts.