Description:

Title : Development and Implementation of an Inducible Type I-C CRISPR-interference system in Neisseria gonorrhoeae

Wendy Geselwitz, Graduate Student, Lab of Dr. Hank Seifert

Faculty Host: Hank Seifert, PhD

Topic:

Gonorrhea is one of the most common sexually transmitted infections worldwide. Neisseria gonorrhoeae (Gc), the sole causative agent of gonorrhea, has grown rapidly antibiotic resistant and we lack a clinically approved vaccine to prevent its spread. Thus, we need new tools to better interrogate Gc function to develop more efficacious therapies against this bacterium. CRISPR systems (Clustered Regularly Interspaced Short Palindromic Repeats) are prokaryotic adaptive immune systems that protect bacteria from potentially harmful foreign DNA and are regularly utilized as DNA editing tools. In addition to generating mutants, CRISPR-Cas has also been repurposed as a gene repression platform known as CRISPR-interference (CRISPRi). With CRISPRi, a nuclease dead Cas protein complex is directed by its CRISPR guide RNA to a specific DNA sequence where it can sterically block RNA polymerase binding and transcription. We have established an IPTG-inducible, CRISPRi system derived from the commensal Neisseria lactamica’s Type I-C CRISPR-Cas for use in Gc. Current work is focused on defining the utility of this tool and fine-tuning its knockdown efficiency. Future application with CRISPRi will allow us to elucidate the function of essential virulence factors via genetic downregulation.