Background: Allergy health problems worldwide are mainly caused by allergens from domestic cats ( Felis catus). Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma.out cats. Fel d 1 level in CH2 knockout cats was assessed by enzyme-linked immunosorbent assay (ELISA). Cytoplasm injection clone technology (CICT) was used to clone the CH2 knockout cat. Results: We report the first successful generation and cloning of CH2 knockout cats using the CRISPR-Cas9 system and CICT. CH2 knockout cats were confirmed using T7E1 and Sanger sequencing. Cloned CH2 knockout cat was verified by microsatellite analysis. Remarkably, ELISA proved that Fel d 1 level of CH2 knockout cats was extremely low compared with that of wild type domestic cats. Conclusion: CH2 knockout cats we generated showed an extremely low level of Fel d 1 and could be hypoallergenic cats. Our study indicates that creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward in the consequence that these cats can safely approach allergic patients.