CRISPRi targeting of Neisseria gonorrhoeae R-loop or G4 mutants restores pilin antigenic variation

Description

Neisseria gonorrhoeae pilin antigenic variation (pilin Av) is a complex diversity generation system. Pilin Av depends on a Rec-dependent gene conversion event initiated by an R-loop (gar sRNA) and a resultant G-quadruplex (G4) DNA structure. Mutations of the garP promoter or the G4-forming sequence each prevent pilin Av. We targeted a Type I-C CRISPR interference complex (CRISPRi) complex to the mutant garP or G4 loci, and CRISPRi targeting to a specific location on the leading strand restored pilin Av. In contrast, targeting the CRISPRi complex to other locations on either the leading or lagging strand was lethal. The CRISPRi restoration of pilin Av depended on the standard recombination factors, confirming the conserved pathway. Inverting the garP-G4 region confirmed that the target strand dictates viability and Av outcome. Together, these results reveal that we can replace the R-loop and resultant G4 structure by specific CRISPRi targeting, providing insights into nucleic acid, secondary structure-dependent genome dynamics.

Additional details