Sequential CRISPR-EspCas9-Mediated Wild-Type Depletion Enhances the Detection Sensitivity of Rare Mutations for Canine Liquid Biopsy Application

One of the major obstacles in early cancer detection in dogs is the limited sensitivity in detecting circulating tumor DNAs (ctDNAs) with low abundances. Standard next-generation sequencing (NGS) without error correction typically achieves detection limits around ~1% mutant allele frequency (MAF). We sought to improve the detection sensitivity using a sequential CRISPR-EspCas9 enrichment strategy in which iterative in vitro cleavage (IVC) was combined with PCR amplification to selectively deplete wild-type DNA and enrich rare tumor mutations. Applying the strategy to genomic DNA and cell-free DNA mimics from canine mammary gland tumor cell lines demonstrated that IVC enrichment enabled the detection of cancer-associated PIK3CA H1047R mutations that were undetectable by conventional Sanger sequencing. To evaluate detection sensitivity, we characterized enrichment using synthetic templates for PIK3CA H1047R and other cancer-related mutations, BRAF V596E, and KRAS G12C. We observed that three iterations of sequential IVC achieved ~160, ~15, and ~2.2-fold enrichment for PIK3CA H1047R, BRAF V596E, and KRAS G12C, respectively. Under the present synthetic-template conditions, the analytical LOD reached 0.001% MAF for PIK3CA and 0.01% MAF for BRAF, whereas KRAS showed only modest enrichment and remained practically limited under the current guide design. Together, the results show that the CRISPR-EspCas9 IVC strategy enables selective enrichment of low-frequency single-nucleotide mutant alleles. We anticipate that the finding could be utilized to develop a highly sensitive veterinary liquid biopsy application with further optimization and validation using canine plasma cfDNA.