PathoGD, an automated bioinformatic pipeline, successfully designed highly specific RPA primers and gRNAs for five bacterial pathogens, demonstrating high specificity with minimal off-target signal.
PathoGD provides an automated, scalable bioinformatic pipeline for the rapid design of highly specific primers and guide RNAs for CRISPR-based pathogen diagnostics.
Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .
Low et al. (Thu,) conducted a other in Bacterial pathogens. PathoGD pipeline was evaluated on Identification and validation of target-specific gRNAs and primers. PathoGD, an automated bioinformatic pipeline, successfully designed highly specific RPA primers and gRNAs for five bacterial pathogens, demonstrating high specificity with minimal off-target signal.