CRISPRi targeting to the Neisseria gonorrhoeae R-loop or G4 mutant region restores pilin antigenic variation

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 (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.IMPORTANCENeisseria's pilin antigenic variation remains one of the most sophisticated DNA diversification processes in bacteria, yet the trigger that initiates recombination remains unclear. Here, we show that targeting a Type I CRISPR interference complex is sufficient to restore pilin variation in strains unable to form an R-loop or a G-quadruplex structure. Our findings reveal that the position and strand of R-loop formation, rather than its precise sequence context, determine whether cells undergo antigenic variation, remain viable, or die. This establishes a new framework in which localized topological stress, not a particular DNA structure, plays a role in Neisseria gonorrhoeae pilin antigenic variation.