(PL23) DEVELOPMENT OF A CRISPR-BASED MULTIPLEX ASSAY FOR NEISSERIA GONORRHOEAE DETECTION AND GYRASE A GENOTYPE DETERMINATION

Background:
Low-resource settings suffer disproportionately from Neisseria (N.) gonorrhoeae infection, but often lack the capacity to support etiologic diagnosis. Syndromic management, however, misses a high proportion of cases and contributes to the development of antimicrobial resistance – a global public health threat. Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) using CRISPR associated (Cas) enzymes and isothermal amplification can facilitate low-cost point-of-care assays. We aimed to develop a multiplex SHERLOCK assay for N. gonorrhoeae detection and predicting ciprofloxacin resistance via identification of a single mutation in the gyrase A (gyrA) gene.

Methods:
We used machine-learning platforms that predict optimal CRISPR guide RNA (crRNA) sequences to develop crRNAs targeting the porA gene for N. gonorrhoeae detection and gyrA S91F. We used Cas12a for porA detection, and Cas13a for gyrA genotype determination. Upon Cas12a crRNA binding, cleavage of a DNA linker releases a HEX reporter, whereas upon Cas13a crRNA binding, cleavage of an RNA linker releases a FAM reporter. We combined the two assays into a single reaction, and evaluated the performance on 10 purified N. gonorrhoeae isolates (2 susceptible to ciprofloxacin with minimum inhibitory concentrations (MIC) <0.015, and 8 resistant with MICs between 8 and > 16). We further performed confirmatory porA detection and gyrA genotyping by qPCR.

Results:
The multiplex CRISPR-based assay identified all 13 isolates and correctly classified all gyrA genotypes as either mutant or non-mutated. Results were available within 90 minutes. The agreement of both CRISPR N. gonorrhoeae detection and gyrA genotype determination with qPCR was 100%.

Conclusion:
We developed a multiplex assay using two Cas orthologs for simultaneous N. gonorrhoeae detection and gyrA genotype determination, which demonstrated promising preliminary performance on purified isolates. Future work can optimize assay kinetics and substitute antigen-bound reporters for quenched fluorophores to produce a field-deployable, multiplexed lateral flow test.