CRISPR/Cas9-mediated Gene Editing of Fel d1 in Cat (Felis catus)
ZHANG Ya-Xin1,*, LI Ling1,2,*, Farhab MUHAMMAD1,2, LYU Mei-Yun1,2, KYAW Paing OO1,2, CAI He-Qing1,2, YUAN Yu-Guo1,2,**
1 College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering, Yangzhou University, Yangzhou 225009, China; 2 Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
Abstract：The major cat (Felis catus) allergen, Fel d1 is composed of 2 heterodimers: chain 1 (CH1) and chain 2 (CH2). Fel d1 could induce allergic reactions to humans. In this study, the genome sequences of CH1 and CH2 of 38 domestic cats of different breeds were analyzed by bioinformatics, and 2 single-guide RNAs (sgRNAs) were designed at the exon 2 of CH2, then the constructed target vector of CRISPR/Cas9 was transfected into the cat fetal fibroblasts cell. The mutation efficiency of the CH2 was verified by PCR and Sanger sequencing. The results showed that the CH1 and CH2 sequences contained 12 and 51 polymorphic loci, and most of these loci were located on the GC-rich of intron 2, and some of them were located on exon 2, intron 3 and exon 3. In the overall evolution, CH1 was more conservative than CH2. The gene editing efficiency of CH2 was totally 40%. There were 2 types of mutation in CH2, which eliminated potential antigen sites. The gene editing efficiency of type 1 with deletion of 45 bases was 35%, and the gene editing efficiency of the type 2 with deletion of 44 bases was 5%. This study provides a way for further use of the target vector to obtain Fel d1 gene mutant cell lines for somatic cell nuclear transfer or embryo injection to obtain hypoallergenic cats.
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