Design guide for synthetic small regulatory RNAs for high-efficiency gene knockdown in bacteria

Small regulatory RNAs (sRNAs) are short noncoding RNAs that can fine-control the expression of target genes in trans at the post-transcriptional level in prokaryotes. Since there is a big challenge in constructing gene-knockout libraries, synthetic sRNAs have attracted considerable interest in synthetic biology and metabolic engineering, as they enable targeted gene knockdown without requiring chromosomal modifications. However, the development of high-efficiency synthetic sRNAs remains a demanding task that requires careful consideration of multiple design factors. Here, we provide a detailed protocol for the design and construction of synthetic sRNAs, detailing key design principles and critical optimization factors, including scaffold selection, target mRNA binding affinity, target mRNA secondary structure, and Hfq expression levels. This strategy can be broadly applied across E. coli and other bacterial hosts to modulate gene expression, thereby supporting versatile applications in synthetic biology and metabolic engineering.