文章摘要
王仿竹,陈红全,王建瑛,沈 彬.应用CRISPR/Cas9和Cas9⁃D10A建立Nsun5基因敲除小鼠模型并分析脱靶效应[J].南京医科大学学报,2020,(9):1275~1280
应用CRISPR/Cas9和Cas9⁃D10A建立Nsun5基因敲除小鼠模型并分析脱靶效应
Generating Nsun5 konckout mice using CRISPR/Cas9 and Cas9⁃D10A system and analyzing the off⁃target effects
投稿时间:2020-06-28  
DOI:10.7655/NYDXBNS20200906
中文关键词: Nsun5  CRISPR/Cas9  基因敲除  脱靶效应
英文关键词: Nsun5  CRISPR/Cas9  gene knockout  off⁃target
基金项目:国家自然科学基金(31970796);江苏省自然科学基金(BK20160045)
作者单位
王仿竹 南京医科大学生殖医学国家重点实验室江苏 南京 211166 
陈红全 浙江大学医学院附属邵逸夫医院检验科浙江 杭州 310016 
王建瑛 南京医科大学生殖医学国家重点实验室江苏 南京 211166 
沈 彬 南京医科大学生殖医学国家重点实验室江苏 南京 211166 
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中文摘要:
      目的:为了检测CRISPR/Cas9系统的潜在脱靶效应,分别利用野生型Cas9和切口酶Cas9?D10A构建了RNA甲基转移酶Nsun5基因敲除小鼠模型,并对两种策略得到的F0小鼠进行脱靶分析,以比较两种敲除策略的特异性。方法:针对Nsun5基因第3外显子,设计1对sgRNA,将野生型Cas9 mRNA和切口酶Cas9?D10A mRNA分别同这对sgRNA混合后注入小鼠一细胞期受精卵中,实现靶基因敲除,比较两种Cas9得到的F0代敲除小鼠的脱靶效应。结果:利用CRISPR/Cas9和Cas9?D10A成功建立Nsun5基因敲除小鼠模型,对两种Cas9得到的F0代突变小鼠进行潜在脱靶位点分析,均未检测到脱靶位点的存在。结论:两种策略都成功构建了不含脱靶位点的Nsun5基因敲除小鼠模型,为进一步研究Nsun5的生物学功能打下基础。
英文摘要:
      Objective:In order to detect the putative off?target effects of CRISPR/Cas9 system,the knockout mouse model of RNA methyltransferase Nsun5 gene were constructed using wild?type Cas9 and nickase Cas9?D10A,respectively,and performed off?target analysis of F0 mice to compare the specificity of the two strategies. Methods:Paired sgRNAs targeting the third exon of Nsun5 were designed and co?injected with Cas9 mRNA and Cas9?D10A mRNA respectively into mouse one?cell fertilized eggs to generate Nsun5 knockout founders. Off?target effects were analyzed in founder mice injected with CRISPR/Cas9 and Cas9?D10A,respectively. Results:CRISPR/Cas9 and Cas9?D10A were used to establish the Nsun5 gene knockout mouse model successfully. The founder mice obtained from the two variants were subject to off?target analyzation,and no off?target sites were detected. Conclusion:Nsun5 gene knockout models were successfully generated by the both strategies without off?target sites,paving the way for further studying the biological functions of Nsun5.
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