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

Abstract

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

Keywords: CRISPR–Cas9; Fel d1; cat; gene knock-out; polymorphism.

Figure 1

Experiment design. DNA samples from 38 domestic cats were collected and amplified by PCR to obtain the complete sequence of the Fel d1 gene. We targeted CH2 and designed two single-guide RNAs for this region, and incorporated these sgRNAs into the PX458 vector, which was used to perform gene knock-out in fetal fibroblasts. It resulted 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 mutation efficiency, physicochemical properties, antigenic sites, and three-dimensional structure of the mutated protein.

Figure 2

Electropherogram of CH1 and CH2 genes from different cat blood DNA samples. (A) Before PCR optimization. Lanes 01–10: CH1 gene, only lane 1 showsa band corresponding to the target size; lanes 11–19: CH2 gene, only lane 11 shows a band corresponding to the target size. (B) After PCR optimization. All lanes show a band corresponding to the target size. Lanes 01–10: CH1 gene; lanes 11–20: CH2 gene; lane 21: negative control; bp: base pairs;and M: DL5000 DNA Marker.

Figure 3

Evolutionary map of the (A) CH1 gene and (B) CH2 gene in different domestic cats.

Figure 4

Schematic diagram of CH2 genes depicting the amplified and sequenced gene regions. (A) The Fel d1 sequence was retrieved from https://www.ncbi.nlm.nih.gov/protein/NP_001041619.1. (accessed on 10 September 2024) Single-underlined nucleotide sequences represent exon 2. The black underline indicates exon 2 of CH2. The red underline indicates target sgRNA candidates within exon 2, the green nucleotides indicate PAM sequences within exon 2, and the red nucleotides indicate sgRNA candidates. The complement to the left-side green nucleotides is CH2-sgRNA-2 (PAM: tca) and the complement to the right-side green nucleotides is CH2-sgRNA-2 (PAM: agg). (B) CH2 gene targeting site. (C) Sanger sequencing peaks of CH2-sgRNA-1. (D) Sanger sequencing peaks of CH2-sgRNA-2. (E) Map of the constructed PX458-CH2-sgRNA plasmids. (F) Mutations within the target sequences compared to the wild type.

Figure 5

(A) Aborted fetus of a domestic cat; (B) domestic cat fetal fibroblasts cultured from an aborted fetus. Fluorescence was measured 36 h after transfection from (C) bright-field microscopy and (D) fluorescent microscopy.

Figure 6

Indels, their frequency (F), prediction analysis diagram of the antigenic sites, and three-dimensional structure of the wild-type and mutant Fel d1 encoded protein. The surface probability plot (Emini) detects the regions of high surface accessibility, and these regions would be potential surface-exposed regions of the protein. The wild-type (WT) protein has an antigenic site at amino acids 53–58. The Type 1 mutation has no significant antigenic sites. The Type 2 mutation has antigenic sites at amino acids 53–57 and 64–69 (created through DNAStar Protean).

Figure 6

Indels, their frequency (F), prediction analysis diagram of the antigenic sites, and three-dimensional structure of the wild-type and mutant Fel d1 encoded protein. The surface probability plot (Emini) detects the regions of high surface accessibility, and these regions would be potential surface-exposed regions of the protein. The wild-type (WT) protein has an antigenic site at amino acids 53–58. The Type 1 mutation has no significant antigenic sites. The Type 2 mutation has antigenic sites at amino acids 53–57 and 64–69 (created through DNAStar Protean).

Figure 7

Analysis of the sequencing results of 20 single clones showed that 12 clones had no mutations (frequency = 60%); 7 clones were missing 45 bases near the target site (frequency 35%); and 1 clone was missing 44 bases near the target site (frequency 5%). (A) Three-dimensional (3D) structure of the encoded protein. Template: P30440.1. A Major allergen I polypeptide chain 2 AlphaFold DB model of FEL1B_FELCA (gene: CH2, organism: Felis silvestris catus). In the Ramachandran Plot, wild type and Type 1 have stable structures, but Type 2 represents two amino acids falling in regions of unusual structural features, i.e., one blue dot representsamino acid 61 within the right bottom quartile, and one red dot representsamino acid 57 within the right-handed-alfa-helix quartile (Created through https://swissmodel.expasy.org/interactive/ (accessed on 10 September 2024)). (B) Comparison of the amino acid precursor sequence of gene-edited Fel d1 (CH2) (5′ → 3′).