Polymorphism Analysis And Gene Editing Of The Feline Fel D1 Gene

Domestic cats are the main allergens in the indoor environment,and the incidence of allergic respiratory diseases caused by them ranks second only to dust mites.Fel dl protein is the most important allergen among the many allergen proteins secreted by cats,secreted by the sebum,glands and salivary glands of cats and present in the skin of cats,causing allergic diseases such as rhinitis and asthma and other respiratory diseases,which can progress to lifethreatening.The rate of cat allergy in the general population is increasing year by year and can reach 10-30%.Fel d1 is a secreted globulin about 35-40 kDa in size,composed of two 18-20 kDa heterodimers,encoded by CH1 and CH2 genes,respectively,using CRISPR technology to genetically edit Fel d1 protein to breed hypoallergenic breed cats,providing a reasonable solution for groups eager to raise cats and prone to allergies.In this study,DNA of 38 domestic cats was collected and amplified by PCR to obtain the full sequence of Fel d1 gene.According to the sequencing results,the polymorphic sites(SNP),insertion/deletion(InDel)sites,nucleotide diversity(Pi),mean nucleotide difference(k)and potential antigenic sites on the sequence were analyzed.CRISPR/Cas9 was used to edit the gene of fetal fibroblasts,and sgRNA was designed according to the conserved site and potential antigenic site of Fel d1 CH2,which was ligated to PX458 plasmid vector.Two knockout plasmids(PX458-CH2-sgRNA-1 and PX458-CH2-sgRNA-2)were successfully constructed.The plasmids were transfected into fetal fibroblasts,the DNA was extracted,the target site product TA was cloned by PCR,and a single colony was sequenced to analyze the changes of physicochemical properties,antigenic site and three-dimensional structure of the mutated protein.The results showed that there were 12 and 51 polymorphic loci(SNP)in CH1 and CH2 sequences respectively,and most of these loci were located in GC-rich Intron 2,while others were located in Exon 2,Intron 3 and Exon 3.In the whole evolution,CH1 is much more conservative than CH2.Two sgRNA were designed to obtain cat fetal fibroblasts with two target gene mutations by using double CRISPR/Cas9 system.The gene editing efficiency of this double CRISPR system was 40%,of which 35%knocked out 45 bases of type 1 mutation,and successfully knocked out potential antigenic sites in CH2;Type 2 mutations that knock out 44 bases account for 5%,knocking out potential antigenic sites,but frameshift mutations may affect other biological functions of Fel d1 protein.In this study,we show that there were 12 polymorphic loci in CH1 and 51 polymorphic loci in CH2,and CH1 was much more conserved than CH2,which provided a reference for understanding the evolution of Fel d1 protein.At the same time,the antigenicity of Fel d1 CH2 gene in domestic cats was reduced by editing CRISPR/Cas9 technology,which laid a foundation for knockout antigenic sites on embryos in the later stage and preparation of hypoallergic cats.