利用CRISPR/Cas9基因编辑技术建立FcγR基因大片段敲除小鼠模型  

Establishment of a large fragment FcγR gene knockout mouse model using CRISPR/Cas9 genome editing technique

作　　者：吴曦[1] 霍桂桃[2] 刘甦苏[1] 谷文达[1] 曹愿 柳全明 吕建军[2] 范昌发[1] WU Xi;HUO Guitao;LIU Susu;GU Wenda;CAO Yuan;LIU Quanming;LYU Jianjun;FAN Changfa(National Center for Laboratory Rodents,National Institutes for Foodand Drug Control,Beijing 102629,China;National Center for Safety Evaluation of Drugs,NationalInstitutes for Food and Drug Control,Beijing 100176)

机构地区：[1]中国食品药品检定研究院实验动物资源研究所,国家啮齿类实验动物种子中心,北京102629 [2]中国食品药品检定研究院国家药物安全评价监测中心,北京100176

出　　处：《中国实验动物学报》2019年第5期583-591,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(81502396);艾滋病和毒性肝炎等重大传染病防治专项(2017ZX10304402)~~

摘　　要：目的使用CRISPR/Cas9基因编辑技术敲除长度约为90kb的小鼠FcγR2b,FcγR3,FcγR4基因簇,为构建FcγR基因人源化小鼠奠定基础。方法使用在线预测软件在FcγR2b,FcγR3外显子区设计sgRNA,在每一位点挑选脱靶效应较低的五个候选sgRNA。通过CRISPR/Cas9活性检测试剂盒检测sgRNA在体外的活性。选取活性较高的sgRNA体外转录,与Cas9mRNA一并注射受精卵。通过PCR检测及测序,得到1只敲除片段为89711bp的基因修饰小鼠,且同时敲除FcγR2b基因5’端,FcγR3基因3’端及FcγR4基因。而且还利用软件预测了8个脱靶可能性最高的位点,并对首建鼠基因组的上述8个脱靶位点全部测序确认。结果结果显示未在预测脱靶位点附近发现小片段插入或缺失。结论建立了利用CRISPR/Cas9基因编辑技术敲除基因组超大片段的技术,该技术结合BAC转基因技术,将为建立含有复杂基因族的人源化小鼠提供新的途径。Objective The mouse FcγR 2 b,FcγR 3,and FcγR 4 gene clusters of about 90 kb in length were knocked out by CRISPR/Cas9 genome editing technique to lay the foundation for constructing an FcγR gene humanized mouse.Methods Single guide RNAs (sgRNAs) were designed using online software to target exons of FcγR 2 b and FcγR 3.Five candidate sgRNAs with lower off-target effects were selected for each target site.The cleavage activity of sgRNAs was tested in vitro by the CRISPR/Cas9 activity assay kit.The sgRNA with high cleavage activity was selected for in vitro transcription and microinjected with Cas9 mRNA into zygotes.Results A genetic modified mouse with 89,711 bp knockout fragment was confirmed by PCR detection and sequencing.The 5′ end of the FcγR 2 b gene,the 3′ end of the FcγR 3 gene and FcγR 4 gene were knocked out at the same time.Moreover,8 sites with the highest probability of off-target were predicted by software, and all 8 off-target sites of the founder mouse genome were sequenced and confirmed.No small fragment insertion or deletion was found in any predicted off-target sites. Conclusions We established a large fragment knockout technique using CRISPR/Cas9 genome editing technique,which may provide a new method to establish humanized mouse models by combining with BAC transgenic strategies.

关 键 词：CRISPR/Cas9 FcγR基因 大片段敲除 小鼠 

分 类 号：Q95-33[生物学—动物学]