Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system

Abstract

Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.

Keywords: Allergen; CH2; CRISPR-Cas9; Fel d 1; Genome-edited cats; Hypoallergenic cats.

Figure 1

Generation and cloning of CH2 genome-edited cats using the CRISPR-Cas9 system and Sanger sequencing. (a) The CH2 genome-edited founder cat (Male, KS-M-001, Heavy). (b) The CH2 genome-edited founder cat (Female, KS-F-011, Haemi). (c) The CH2 genome-edited cat (F1, Male, CH2^−/−, M-008, Alsik). (d) The CH2 genome-edited cloned cat (Male, CH2^-/-, KS-M-004, Alsik C) from “Alsik”. (e) The modified target region in each CH2 genome-edited cat. (f) The sequencing chromatogram of the target region of the CH2 genome-edited founder cat (Heavy). (g) The sequencing chromatogram of the target region of the CH2 genome-edited founder cat (Haemi). (h) The sequencing chromatogram of the target region of the CH2 genome-edited cat (Alsik). (i) The sequencing chromatogram of the target region of the CH2 genome-edited cloned cat (Alsik C). “Heavy” and “Haemi” were generated by cytoplasmic microinjection of sgRNA C2-1 and Cas9 mRNA and embryo transfer. “Alsik” was generated from the mating of “Heavy” and “Haemi”. “Alsik C” was cloned from “Alsik” via CICT. Through the use of PCR and Sanger sequencing, mutations are found in every CH2 genome-edited cat. The blue highlights indicate the Cas9-targeted CH2 sequences. The PAM sequence is highlighted in bold blue. Bold red is used to highlight the indels. Each CH2 genome-edited cat’s DNA sequence is in alignment with the WT cat’s DNA sequence. CICT; cytoplasm injection cloning technology. ID; identification. CH2; Fel d 1 chain 2. Indel; insertion/deletion. PAM; protospacer adjacent motif. WT; wild type. Ns; nucleotides. T; thymine. G; guanine.

Figure 1

Generation and cloning of CH2 genome-edited cats using the CRISPR-Cas9 system and Sanger sequencing. (a) The CH2 genome-edited founder cat (Male, KS-M-001, Heavy). (b) The CH2 genome-edited founder cat (Female, KS-F-011, Haemi). (c) The CH2 genome-edited cat (F1, Male, CH2^−/−, M-008, Alsik). (d) The CH2 genome-edited cloned cat (Male, CH2^-/-, KS-M-004, Alsik C) from “Alsik”. (e) The modified target region in each CH2 genome-edited cat. (f) The sequencing chromatogram of the target region of the CH2 genome-edited founder cat (Heavy). (g) The sequencing chromatogram of the target region of the CH2 genome-edited founder cat (Haemi). (h) The sequencing chromatogram of the target region of the CH2 genome-edited cat (Alsik). (i) The sequencing chromatogram of the target region of the CH2 genome-edited cloned cat (Alsik C). “Heavy” and “Haemi” were generated by cytoplasmic microinjection of sgRNA C2-1 and Cas9 mRNA and embryo transfer. “Alsik” was generated from the mating of “Heavy” and “Haemi”. “Alsik C” was cloned from “Alsik” via CICT. Through the use of PCR and Sanger sequencing, mutations are found in every CH2 genome-edited cat. The blue highlights indicate the Cas9-targeted CH2 sequences. The PAM sequence is highlighted in bold blue. Bold red is used to highlight the indels. Each CH2 genome-edited cat’s DNA sequence is in alignment with the WT cat’s DNA sequence. CICT; cytoplasm injection cloning technology. ID; identification. CH2; Fel d 1 chain 2. Indel; insertion/deletion. PAM; protospacer adjacent motif. WT; wild type. Ns; nucleotides. T; thymine. G; guanine.

Figure 2

Production level of Fel d 1 in CH2 genome-edited cats before- and after washing. (a) Fel d 1 levels in the saliva of CH2 genome-edited male cats. (b) Fel d 1 levels in the saliva of CH2 genome-edited female cats. (c) Fel d 1 levels in the fur of CH2 genome-edited male cats. (d) Fel d 1 levels in the fur of CH2 genome-edited female cats. The production level of Fel d 1 was expressed as mean with standard deviation. The statistical significance of each value is presented in Supplementary Table 8. W; washing. W-1; the day before washing. W + 1; the day after washing. W + 4; day 4 after washing. W + 7; day 7 after washing.