EXCAVATE-HT: A Bioinformatic Pipeline to Identify Targetable Genomic Variants for Allele-Specific Editing

Area of Science:

• Genetics
• Bioinformatics
• Molecular Biology

Background:

• Allele-specific CRISPR/Cas editing offers a promising therapeutic strategy for genetic diseases.
• Targeting common single nucleotide polymorphisms (SNPs) can broaden treatment applicability compared to rare mutations.
• Existing tools for variant identification and guide RNA generation have limitations.

Purpose of the Study:

• To introduce EXCAVATE-HT, a novel bioinformatics tool for designing CRISPR libraries targeting common variants for allele-specific editing.
• To enable high-throughput identification of targetable SNPs and generation of allele-specific guide RNAs (gRNAs).

Main Methods:

• EXCAVATE-HT mines population variant data to identify common SNPs within user-defined genomic loci.
• The tool generates annotated lists of allele-specific gRNAs and gRNA pairs for targeted editing or excision.
• Designed and characterized gRNA libraries for allele-specific targeting of the CRX gene.

Main Results:

• EXCAVATE-HT successfully identified multiple allele-specific gRNAs for targeting the CRX gene.
• The developed gRNA libraries demonstrated potential for allele-specific editing.
• Identified excisions could potentially treat over 30-fold more patients than targeting a single CRX disease mutation.

Conclusions:

• EXCAVATE-HT is an effective tool for designing CRISPR libraries for allele-specific editing.
• This approach significantly expands the potential patient population for genetic disease therapies.
• EXCAVATE-HT addresses limitations in current CRISPR tool development for common variant targeting.