TY - JOUR
T1 - PathoGD
T2 - an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics
AU - Low, Soo Jen
AU - O'Neill, Matthew
AU - Kerry, William J
AU - Wild, Natasha
AU - Krysiak, Marcelina
AU - Nong, Yi
AU - Azzato, Francesca
AU - Hor, Eileen
AU - Williams, Lewis
AU - Taiaroa, George
AU - Steinig, Eike
AU - Pasricha, Shivani
AU - Williamson, Deborah A
N1 - Funding: This study was funded by the Victorian Medical Research Accelerator Fund (GA-F3791196–5514) and the Australian Government Department of Health (PO4932). D.A.W. is supported by an NHMRC Investigator Grant (APP1174555). This work was also supported by an Australian Research Council Industrial Transformation Research Hub Grant (IH190100021).
PY - 2025/1/30
Y1 - 2025/1/30
N2 - 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 .
AB - 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 .
KW - Genomics/methods
KW - Streptococcus pyogenes/genetics
KW - CRISPR-Cas systems
KW - RNA, Guide, CRISPR-Cas systems/genetics
KW - Neisseria gonorrhoeae/genetics
KW - Humans
KW - DNA primers/genetics
KW - Computational biology/methods
KW - Clustered regularly interspaced short palindromic repeats
U2 - 10.1038/s42003-025-07591-1
DO - 10.1038/s42003-025-07591-1
M3 - Article
C2 - 39885339
SN - 2399-3642
VL - 8
SP - 1
EP - 13
JO - Communications Biology
JF - Communications Biology
M1 - 147
ER -