EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• CRISPR-Cas9 genome editing offers versatile genetic manipulation across organisms.
• Adapting CRISPR-Cas9 to eukaryotic pathogens facilitates research on these challenging organisms.

Purpose of the Study:

• To introduce the Eukaryotic Pathogen gRNA Design Tool (EuPaGDT), a web-based resource for designing guide RNA (gRNA) for CRISPR-Cas9 applications in eukaryotic pathogens.
• To provide a flexible and high-throughput gRNA design solution tailored to the unique genetic attributes of eukaryotic pathogens.

Main Methods:

• Development of the EuPaGDT webtool (http://grna.ctegd.uga.edu) for automated gRNA identification and scoring.
• Implementation of an on-target search algorithm to identify gRNAs targeting multi-gene families.
• Inclusion of scoring for microhomology sequences flanking gRNA cut sites to aid in double-strand break repair analysis.
• Support for designing single-stranded oligonucleotides for homology-directed repair.
• Batch processing capability for genome-scale sequence analysis and gRNA library preparation.

Main Results:

• EuPaGDT successfully identifies and ranks potential gRNAs for eukaryotic pathogens, considering unique genomic features.
• The tool effectively targets multi-gene families, crucial for understanding host-pathogen interactions.
• EuPaGDT facilitates the design of gRNAs and associated repair templates for efficient genome editing in pathogens.
• Batch processing enables large-scale gRNA library design for high-throughput screening.

Conclusions:

• EuPaGDT is a valuable, specialized tool for advancing CRISPR-Cas9 based research in eukaryotic pathogens.
• The webtool enhances the ease and efficiency of gRNA design, supporting complex genetic studies and large-scale screening projects.