Targeted Gene Knock-Out of in Fetal Fibroblasts Using CRISPR-Cas9: Implications for Cat Allergies.

is the most important allergen secreted by cats, which can trigger asthma in sensitive individuals. Our objective was to knock-out the gene in the fetal fibroblasts of cats through CRISPR-Cas9 technology with two sgRNAs and to determine the impact of such mutations on the antigenicity of the protein. DNA samples from 38 domestic cats were collected and amplified by PCR to obtain the complete sequence of the gene. Throughout evolution, polypeptide chain 1() has proven to be much more conserved than polypeptide chain 2(); therefore, we targeted and designed two single-guide RNAs (-sgRNA-1 and -sgRNA-2) for this region. Using these constructed sgRNAs, we performed gene knock-out in fetal fibroblasts, resulting in two mutations within the target gene. Following this, DNA was extracted and the target site product was cloned using TA cloning via PCR, and a single colony from this process was sequenced to analyze the physicochemical properties, antigenic sites, and three-dimensional structure of the mutated protein. The results revealed that there were 12 and 51 polymorphic loci (single-nucleotide polymorphisms, or SNPs) found in the and sequences, respectively, with most loci located in the GC-rich intron 2, while others were found in exon 2, intron 3, and exon 3. These SNPs guided sgRNA design by identifying conserved regions in the gene. The gene editing efficiency for the region, with this dual CRISPR system, was 40%, with 35% attributed to Type 1 mutation and 5% to Type 2 mutation. In conclusion, is significantly more conserved than , and the antigenicity of the gene in domestic cats can be effectively reduced through CRISPR-Cas9 gene editing.