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| 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 |
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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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Received: 31 July 2023
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Corresponding Authors:
** yyg9776430@163.com
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| About author:: * These authors contributed equally to this work |
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[1] 马宇浩, 高爽, 董向会, 等. 2020. 基因编辑在农业动物中的应用进展[J]. 农业生物技术学报, 8(12): 2230~2239.
(Ma Y Hao, Gao S, Dong X H, et al.2020. Application progress of gene editing in agricultural animals[J]. Journal of Agricultural Biotechnology, 28(12): 2230~2239.)
[2] 裴业春, 安晓荣, 侯健, 等. 2016. 猫重组变应原Fel d1与乙肝病毒核心抗原融合基因的原核表达[J]. 中国畜牧兽医, 43(11): 2826-2833.
(Pei Y C, An X L, Hou J, et al.2016. Prokaryotic expression of fusion gene of cat recombinant allergen fel d1 with Hepatitis BC core antigen[J]. China Animal Husbandry & Veterinary Medicine, 43(11): 2826-2833.)
[3] Brackett N F, Pomés A, Chapman M D.2022a. New frontiers: Precise editing of allergen genes using CRISPR[J]. Frontiers in Allergy, 2: 821107.
[4] Brackett N F, Davis B W, Adli M, et al.2022b. Evolutionary biology and gene editing of cat allergen, fel d1[J]. The CRISPR Journal, 5(2): 213-223.
[5] Brackett N F, Pomés A, Allergy M C, et al.2021. The major cat allergen, fel d1, is a viable target for CRISPR gene editing[J]. The CRISPR Journal, 147(2): 175-182.
[6] Cho S J, Bang J I, Yu X F, et al.2010. Generation of a recloned transgenic cat expressing red fluorescence protein[J]. Theriogenology, 73: 848-855.
[7] Chung A G, Belone P M, Bímová B V, et al.2017. Studies of an androgen-binding protein knockout corroborate a role for salivary ABP in mouse communication[J]. Genetics, 205(4): 1517-1527.
[8] Curin M, Weber M, Thalhamer T, et al.2014. Hypoallergenic derivatives of fel d1 obtained by rational reassembly for allergy vaccination and tolerance induction[J]. Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology, 44(6): 882-894.
[9] Dance A.2020. The race to deliver the hypoallergenic cat[J]. Nature, 588(7836): S7-S9.
[10] Griffith I J, Craig S, Pollock J, et al.1992. Expression and genomic structure of the genes encoding FdI, the major allergen from the domestic cat[J]. Gene, 113(2): 263-268.
[11] Jiang T, Zhang X O, Weng Z, et al.2022. Deletion and replacement of long genomic sequences using prime editing[J]. Nature Biotechnology, 40(2): 227-234.
[12] Liu G, Lin Q, Jin S, et al.2022. The CRISPR-Cas toolbox and gene editing technologies[J]. Molecular Cell, 82(2): 333-347.
[13] Lyons L A.2022. It's a knockout for cat allergies?[J]. The CRISPR Journal, 5(3): 356-357.
[14] Morgenstern J P, Griffith I J, Brauer A W, et al.1991. Amino acid sequence of Fel d1, the major allergen of the domestic cat: Protein sequence analysis and cDNA cloning[J]. Proceedings of the National Academy of Sciences of the USA, 88(21): 9690-9694.
[15] Perisse I V, Fan Z, Singina G N, et al.2021. Improvements in gene editing technology boost its applications in livestock[J]. Frontiers in Genetics, 11: 614688.
[16] Riabova K, Karsonova A V, van Hage M, et al.2022. Molecular allergen-specific IgE recognition profiles and cumulative specific IgE levels associated with phenotypes of cat allergy[J]. International Journal of Molecular Sciences, 23(13): 6984.
[17] Saarne T, Kaiser L, Grönlund H, et al.2005. Rational design of hypoallergens applied to the major cat allergen Fel d1[J]. Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology, 35(5): 657-663.
[18] Sartore S, Landoni E, Maione S, et al.2017. Polymorphism analysis of Ch1 and Ch2 genes in the siberian cat[J]. Veterinary Sciences, 4(4): 623-625.
[19] Sparkes A H.2022. Human allergy to cats: A review for veterinarians on prevalence, causes, symptoms and control[J]. International Society of Feline Medicine, 24: 31-42. |
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