PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics

Area of Science:

• Bioinformatics
• Molecular Diagnostics
• Genomics

Background:

• CRISPR-based diagnostics require highly specific target selection for accurate pathogen identification.
• Manual design of diagnostic targets is time-consuming and requires iterative optimization.

Purpose of the Study:

• To develop an automated bioinformatic pipeline, PathoGD, for rapid and high-throughput design of primers and guide RNAs (gRNAs).
• To facilitate the development of CRISPR-Cas12a-based diagnostic assays for detecting clinically relevant bacterial pathogens.

Main Methods:

• PathoGD leverages publicly available genomic sequences for automated primer and gRNA design.
• The pipeline is scalable for large datasets, enabling continuous monitoring and validation of assay components.
• Experimental validation was performed for Streptococcus pyogenes and Neisseria gonorrhoeae detection.

Main Results:

• PathoGD successfully designed primers and gRNAs for five bacterial pathogens.
• Experimental validation confirmed high specificity and minimal off-target signals for designed primer and gRNA sets.
• The pipeline demonstrated efficacy in detecting target pathogens with and without pre-amplification.

Conclusions:

• PathoGD offers a rapid and automated solution for designing CRISPR-Cas12a diagnostic assay components.
• The pipeline ensures ongoing assay relevance through continuous monitoring and validation.
• PathoGD is a valuable resource for developing diagnostic tools for current and emerging pathogens.