PROJECT SUMMARY/ABSTRACT Gonorrhea, caused by the Gram-negative bacteria Neisseria gonorrhoeae (NGO), is one of the most prevalent sexually transmitted infections worldwide. In 2013 and 2019 the CDC’s antibiotic resistance threats reports, designated a threat level of urgent to NGO, in part due to the alarming rise in the number of highly antibiotic resistant strains. Resistance to last line antibiotics and treatment failure is now common and there is a critic need to explore new approaches to develop therapeutic interventions. Small regulatory RNAs (herein called sRNAs) are an important class of regulatory molecules in bacterial cells. sRNAs control numerous cellular processes, and for many bacterial pathogens, sRNAs play a critical role during transmission and infection. Despite their importance sRNAs are extremely understudied (to date, in NGO only 7 sRNA have been studied in detail). One major impediment to sRNA study is the absence of sRNA genes from annotated genome reference files. As a consequence, techniques that rely on genome annotation files (such as RNAseq) omit sRNAs. To address this oversight, and to facilitate improved study of sRNAs in NGO, we have constructed a genome annotation file containing annotations for 559 previously reported potential sRNAs in NGO strain FA1090. Using this file, we have reanalyzed publicly available RNAseq data sets and generated a list of 88 prioritized sRNAs which we consider have high potential to be bona fide trans acting sRNAs in NGO. In this study we propose to construct the first NGO sRNA mutant library in which we generate 88 sRNA mutants by knocking out 88 previously unstudied sRNA genes. We will go on to phenotypically characterize the newly created sRNA mutant library to elucidate role(s) for these molecules.