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 |
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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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| author | Soo Jen Low Matthew O’Neill William J. Kerry Natasha Wild Marcelina Krysiak Yi Nong Francesca Azzato Eileen Hor Lewis Williams George Taiaroa Eike Steinig Shivani Pasricha Deborah A. Williamson |
| author_facet | Soo Jen Low Matthew O’Neill William J. Kerry Natasha Wild Marcelina Krysiak Yi Nong Francesca Azzato Eileen Hor Lewis Williams George Taiaroa Eike Steinig Shivani Pasricha Deborah A. Williamson |
| author_sort | Soo Jen Low |
| collection | DOAJ |
| description | 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 . |
| format | Article |
| id | doaj-art-857c41c3beb7475b826c1c60f51512ed |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-857c41c3beb7475b826c1c60f51512ed2025-02-02T12:37:15ZengNature PortfolioCommunications Biology2399-36422025-01-018111310.1038/s42003-025-07591-1PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnosticsSoo Jen Low0Matthew O’Neill1William J. Kerry2Natasha Wild3Marcelina Krysiak4Yi Nong5Francesca Azzato6Eileen Hor7Lewis Williams8George Taiaroa9Eike Steinig10Shivani Pasricha11Deborah A. Williamson12Department of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityWalter and Eliza Hall Institute of Medical ResearchWalter and Eliza Hall Institute of Medical ResearchDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityVictorian Infectious Diseases Reference Laboratory, The Royal Melbourne Hospital at The Peter Doherty Institute for Infection and ImmunityWalter and Eliza Hall Institute of Medical ResearchDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityDepartment of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and ImmunityAbstract 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 .https://doi.org/10.1038/s42003-025-07591-1 |
| spellingShingle | Soo Jen Low Matthew O’Neill William J. Kerry Natasha Wild Marcelina Krysiak Yi Nong Francesca Azzato Eileen Hor Lewis Williams George Taiaroa Eike Steinig Shivani Pasricha Deborah A. Williamson PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics Communications Biology |
| title | PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics |
| title_full | PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics |
| title_fullStr | PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics |
| title_full_unstemmed | PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics |
| title_short | PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics |
| title_sort | pathogd an integrative genomics approach to primer and guide rna design for crispr based diagnostics |
| url | https://doi.org/10.1038/s42003-025-07591-1 |
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