CRISPR/Cas9基因编辑系统应用于非人灵长类细胞及胚胎Rb1基因编辑的研究  

CRISPR/Cas9 applied to editing nonhuman primate cells and embryos Rb1 suppressor gene

作　　者：怀思远 褚楚 曹静 赵志飞[1] 吴璇[1] 冀天楠 李建雄[1] HUAI Siyuan;CHU Chu;CAO Jing;ZHAO Zhifei;WU Xuan;JI Tiannan;LI Jianxiong(Department of Radiotherapy,Chinese PLA General Hospital,Beijing 100853,China;Yunnan Key Laboratory of Primate Biomedical Research,Kunming 650500,Yunnan Province,China)

机构地区：[1]解放军总医院放射治疗科,北京100853 [2]云南中科灵长类生物医学重点实验室,云南昆明650500

出　　处：《解放军医学院学报》2018年第8期707-712,共6页Academic Journal of Chinese PLA Medical School

摘　　要：目的探索聚簇规则间隔短回文重复序列/CRISPR相关核酸酶9(CRISPR/Cas9)基因编辑系统对于食蟹猴细胞及胚胎基因组视网膜母细胞瘤抑癌基因(Rb1)的编辑效率,旨在进一步建立视网膜母细胞瘤模型。方法查阅RB1突变数据库(http://rb1-lsdb.d-lohmann.de),确定视母细胞瘤发生发展相关Rb1基因外显子区主要有8号外显子;利用DNAman检测人类与食蟹猴基因组中Rb1基因中与是否存在单核苷酸多态性(SNP);通过http://crispr.cos.uni-heidelberg.de/index.html设计并合成sgRNA;采用CRISPR/Cas9基因编辑系统转染食蟹猴成纤维细胞,并提取转染后细胞基因组以检测合成的sgRNA敲除效率;酶切实验检测转染的细胞是否发生突变,并利用单克隆分析测定具体敲除效率。进一步利用显微操作技术注射sgRNA和Cas9 nuclease混合物(RNP)入单细胞胚胎,检测胚胎水平Rb1基因编辑效率。结果 RNP转染后的食蟹猴成纤维细胞提取基因组检测,发现其中有75%的细胞发生基因突变,且基因编辑类型多样,以碱基缺失突变为主;在胚胎水平上获取4-细胞期胚胎,检测单个卵裂球基因组序列发现,存在突变的胚胎占所有胚胎的36.4%,其中纯合突变比例为18.2%,嵌合突变比例同为18.2%。利用软件预测脱靶位点12个,进行序列测定,结果未发现脱靶。结论在非人灵长类细胞及胚胎水平上,CRISPR/Cas9基因编辑系统对于食蟹猴Rb1基因编辑有效,并且敲除效率较高,进一步利用基因编辑方法建立视网膜母细胞瘤模型研究中,可以作为有效的基因编辑手段。本研究得到的食蟹猴胚胎Rb1基因的编辑效率及胚胎囊胚率为建立视网膜母细胞瘤模型提供了参考。Objective To explore the editing efficiency of CRISPR/Cas9 gene editing system for cynomolgus monkey cells and embryos retinoblastoma tumor suppressor gene（Rb1） in order to further establish retinoblastoma model. Methods Rb1 mutation database（http：//rb1-lsdb.d-lohmann.de） was referred to determine exon 8 which was the main exon of Rb1 that related to the incidence and development of retinoblastoma tumor; DNAman was applied to detect whether there was a SNP in the Rb1 gene between human and macaque monkey; Then we use http：//crispr.cos.uni-heidelberg.de/index.html to design the sg RNA sequence; The CRISPR/Cas9 gene editing system was performed to detect the knock-out efficiency by transfecting RNP（sg RNA and Cas9 nuclease mixture） into the cells; Enzyme digestion experiment was used to detect the type of mutations, and monoclonal gene analysis to determine the specific knock-out efficiency. Further microinjection technique was performed to inject RNP into singlecell embryos to detect embryonic Rb1 gene editing efficiency. Results The genome of the RNP-transfected cynomolgus monkey fibroblast cells were extracted, 75% of the cells were found to have gene mutations, and the type of gene editing varied, with basedeleted mutations being dominant; 4-cell stage embryo was obtained and the detection of a single blastomere genome sequence revealed that the mutant embryos accounted for 36.4% of all embryos, of which the homozygous mutation ratio was 18.2% and the chimeric mutation ratio was 18.2%. Twelve off-target sites were predicted using software, and sequencing was performed. As a result, no off-target was found. Conclusion In non-human primate cells and embryos, the CRISPR/Cas9 gene editing system is effective for Rb1 editing of cynomolgus monkeys, and it has a higher knock-out efficiency, which means using gene editing methods to establish a retinoblastoma model can be achieved later. As an effective gene editing tool, the editing efficiency of Rb1 gene and the rate of embryonic blastocyst in the cyn

关 键 词：视网膜母细胞瘤 聚簇规则间隔短回文重复序列/相关核酸酶9 基因敲除 打靶效率 脱靶效率 

分 类 号：R739.72[医药卫生—肿瘤]