CRISPR/Cas9-Mediated CH2 Deletion of Fel d1 Triggers Transcriptomic Reprogramming and Disease-Associated Pathways in Feline Cells

Lin-Yi Qu¹Fu-Shi Quan¹Shu-Ming Shi¹Zhi-Chao Chi¹Yongxun Jin¹Ming-Jun Zhang¹Il-Keun KONG^1,2Xianfeng Yu^1*

• ¹Jilin University, Changchun, China
• ²Gyeongsang National University, Jinju-si, Republic of Korea

Fel d1, the major cat allergen responsible for over 90% of human IgE-mediated allergies, has an unclear physiological role. To explore its function and assess the feasibility of producing hypoallergenic cats, we used CRISPR/Cas9 to knockout the CH2 gene, which encodes one chain of the Fel d1 heterodimer, in domestic cat skin cells. We employed an optimized sgRNA that introduced a three-base insertion, leading to a significant decrease in both CH2 mRNA and Fel d1 protein. Transcriptome sequencing revealed extensive transcriptional reprogramming, identifying 3,469 differentially expressed genes and enrichment in disease-associated pathways, particularly hypertrophic cardiomyopathy (HCM) and rheumatoid arthritis (RA). GO and KEGG analyses indicated alterations in calcium signaling, extracellular matrix remodeling, and immune-related processes. qPCR validation of key genes (including TGFB2, MYBPC3, MMP3, and TLR4) confirmed potential risks of unintended pathological responses. Furthermore, CH2 deletion disrupted transcripts linked to pro-inflammatory signaling and intracellular signal fidelity. These findings highlight the importance of comprehensive transcriptomic evaluation following gene editing and offer critical insights for improving the safety and efficiency of SCNT-based production of hypoallergenic cats.