PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics
Abstract 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 ra...
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| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-07591-1 |
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| Summary: | Abstract 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 . |
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| ISSN: | 2399-3642 |