Development of Cas13a-based Assays for Neisseria gonorrhoeae Detection and Gyrase A Determination

Abstract

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 por A gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A ( gyr A) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For por A, 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 gyr A, 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 gyr A 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.

Figure 1:. Guide and Primer Selection for a Cas13a-based assay for detecting N. Gonorrhoeae

Panel a) shows the performance of three guides targeting different regions of the porA gene tested on 3 N. gonorrhoeae purified isolates as well as synthetic gyrA template as a control and a negative template control (NTC). Panel b) shows the selected porA guide sequence. *** Indicates statistically significant differences in florescence at the p<0.05 level

Figure 2:. In vitro limit of detection of the Cas13a N. gonorrhoeae and gyrA genotypic assays

Panel a) shows the limit of detection of the N. gonorrhoeae Cas13a detection assay using the selected guide-primer set for the porA gene among purified N. gonorrhoeae isolates and a negative template control (NTC); Panel b) shows the_limit of detection of the Cas13a-based assay using the wildtype guide against synthetic wildtype DNA target; Panel c) shows the limit of detection of Cas13a-based assay using the mutant guide against synthetic mutant DNA target. The serial dilutions of synthetic DNA were done in water.

Figure 3:. Performance of a Cas13a-Based Lateral Flow Assay for Detecting N. gonorrhoeae

Panel a) shows the performance of the Cas13a-based lateral flow assay on 14 purified N. gonorrhoeae isolates tested in triplicate. Panel b) shows the discrimination of the lateral flow assay for N. gonorrhoeae isolates compared with non-gonococcal Neisseria isolates. * The faint band at the test line in the negative control is expected per the manufacturer protocol

Figure 4:. Cas13a-Based Gyrase A Determination of Purified N. gonorrhoeae Specimens

The figure shows the pooled discrimination of the Cas13a-based assay using fluorescence detection for determining the gyrA genotype of 23 purified N. gonorrhoeae isolates.

Figure 5:. DNA Sequence Alignment of Codon 91 of the gyrA Gene from 23 Purified N. gonorrhoeae Isolates

The figure shows the DNA sequence alignment of codon 91 of the gyrA gene in N. gonorrhoeae with the two CRISPR-Cas13a guide sequences.

Figure 6:. Performance of a Cas13a-Based gyrA Assay Using Lateral Flow Strips and a Portable Quantitative Fluorometer on Purified N. gonorrhoeae Isolates

Panel a) shows the performance of a Cas13a-based lateral flow assay using both wildtype and mutant guides for determining gyrA genotype among 3 N. gonorrhoeae isolates. Panel b) shows the same Cas13a assay read on a Qubit 4 Fluorometer. NTC (Negative Template Control)