基于CRISPR/Cas9技术进行家猫Fel d1基因编辑的研究  

CRISPR/Cas9-mediated Gene Editing of Fel d1 in Cat(Felis catus)

作　　者：张亚鑫 李玲[1,2] Farhab MUHAMMAD 吕美芸 佰伍加 蔡何清 袁玉国 ZHANG Ya-Xin;LI Ling;Farhab MUHAMMAD;LYU Mei-Yun;KYAW Paing OO;CAI He-Qing;YUAN Yu-Guo(College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering,Yangzhou University,Yangzhou 225009,China;Jiangsu CoInnovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院/扬州大学动物基因工程重点实验室,扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《农业生物技术学报》2024年第6期1353-1361,共9页Journal of Agricultural Biotechnology

基　　金：江苏省种业振兴“揭榜挂帅”项目(JBGS[2021]025);江苏高校优势学科建设工程资助项目(PAPD);高等学校学科创新引智计划(D18007);扬州大学大学生科创基金(X20230665)。

摘　　要：Fel d1蛋白是家猫(Felis catus)过敏原中最主要的致敏原,可引起人不同程度的过敏反应。Fel d1蛋白由2个异源的二聚体组成,其编码基因分别为chain 1(CH1)和chain 2(CH2)。本研究对38只不同品种的家养宠物猫CH1和CH2基因组序列进行生物信息学分析,进一步在CH2外显子位点设计2个单链引导RNA(single-guide RNAs,sgRNAs),构建打靶载体分别转染猫胎儿成纤维细胞,PCR测序验证靶位点突变效率。结果表明,CH1和CH2序列上分别含有12和51个多态性位点,且这些位点大多在富含GC的内含子2上,其他个别位于外显子2,内含子3和外显子3上。在整体的进化演变中,CH1的保守性要远高于CH2。CRISPR/Cas9打靶载体转染猫胎儿成纤维细胞后突变检测表明,CH2基因编辑效率为40%,2种突变方式均能消除潜在的抗原位点,其中敲除45个碱基的1型突变占比35%,敲除44个碱基的2型突变占比5%。本研究为进一步利用该CRISPR/Cas9打靶载体获得Fel d1基因突变细胞株进行体细胞核移植或胚胎注射获得低过敏原猫提供参考。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.

关 键 词：Fel d1基因 CRISPR/Cas9 猫 基因编辑 

分 类 号：S813.3[农业科学—畜牧学] S829.3[农业科学—畜牧兽医]