95 / 2024-10-26 06:50:44

CRISPR/Cas9-Mediated Knockout of Fel d1: Implications for Reducing Allergens in Cats and Enhancing Human Health

Fel d1, the primary allergen linked to domestic cats (Felis catus), consists of two heterodimeric chains, chain1 (CH1) and chain2 (CH2), and is a significant trigger for allergic reactions in sensitized individuals. There is an urgent need for effective strategies to mitigate the impact of this allergen. This study presents a comprehensive bioinformatics analysis of the genomic sequences of CH1 and CH2 from38 domestic cats across various breeds. We designed two single-guide RNAs (sgRNAs) aimed at exon2 of CH2 and successfully transfected the CRISPR/Cas9 target vector into cat fetal fibroblast cells to enable gene editing. The mutation efficiency of CH2 was evaluated through PCR and Sanger sequencing. Our results identified 12 polymorphic loci in CH1 and 51 in CH2, predominantly located in the GC-rich region of intron2, with additional variations found in exon 2, intron 3, and exon 3. CH1 demonstrated greater evolutionary conservation compared to CH2. The overall gene editing efficiency for CH2 reached 40%, resulting in two distinct mutation types that effectively eliminated potential antigenic sites. Type1 mutations, characterized by a deletion of 45 base pairs, achieved a35% editing efficiency, while Type2 mutations, involving a deletion of 44 base pairs, exhibited a 5% efficiency. This research lays the groundwork for generating Fel d1 gene mutant cell lines, which may be employed in somatic cell nuclear transfer or embryo injection, ultimately advancing the development of hypoallergenic cats. By investing in CRISPR/Cas9 gene editing advancements, we aim to enhance the health and happiness of both cats and their owners, fostering a more sustainable approach to managing allergies and addressing concerns related to antibiotic resistance.