Targeted Gene Knock-Out of Fel d1 in Fetal Fibroblasts Using CRISPR–Cas9: Implications for Cat Allergies

<i>Fel d1</i> is the most important allergen secreted by cats, which can trigger asthma in sensitive individuals. Our objective was to knock-out the <i>Fel d1</i> 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 <i>Fel d1</i> protein. DNA samples from 38 domestic cats were collected and amplified by PCR to obtain the complete sequence of the <i>Fel d1</i> gene. Throughout evolution, <i>Fel d1</i> polypeptide chain 1(<i>CH1</i>) has proven to be much more conserved than <i>Fel d1</i> polypeptide chain 2(<i>CH2</i>); therefore, we targeted <i>CH2</i> and designed two single-guide RNAs (<i>CH2</i>-sgRNA-1 and <i>CH2</i>-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 <i>CH1</i> and <i>CH2</i> 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 <i>CH2</i> gene. The gene editing efficiency for the <i>CH2</i> region, with this dual CRISPR system, was 40%, with 35% attributed to Type 1 mutation and 5% to Type 2 mutation. In conclusion, <i>CH1</i> is significantly more conserved than <i>CH2</i>, and the antigenicity of the <i>Fel d1 CH2</i> gene in domestic cats can be effectively reduced through CRISPR-Cas9 gene editing.