Background Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where prevalence is highest. Recent advances in molecular diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. Methods and Results We designed and optimized RNA guides and primer-sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the porA gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A (gyrA) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For porA, we created both a fluorescence-based assay and lateral flow assay using a biotinylated FAM reporter. Both methods demonstrated sensitive detection of 14 N. gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyrA, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyrA genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. Conclusion We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates.

P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, as well as a board member of and shareholder in Danaher Corporation. J.E.L previously served as a consultant to SHERLOCK Biosciences. J.B. has received research funding from Analog Devices Inc., Zeus Scientific, Immunetics, Pfizer, DiaSorin and bioMerieux, and has been a paid consultant to T2 Biosystems, DiaSorin, and Roche Diagnostics. The remaining authors have nothing to disclose.

This work was supported in part by the Massachusetts General Hospital Department of Medicine Innovation Program grant to R.H.G, NIH NIAID U19AI110818 to P.C.S, and grants 2019123 and 2021287 from the Doris Duke Charitable Foundation to J.E.L.

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The Mass General Brigham Institutional Review Board approved this study under protocols 2019P003305 and 2020P000323

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