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。
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.
作者
王仿竹
陈红全
王建瑛
沈彬
WANG Fangzhu;CHEN Hongquan;WANG Jianying;SHEN Bin(State Key Laboratory of Reproductive Medicine,Nanjing Medical University,Nanjing 211166;Department of Laboratory Medicine,Sir Runrun Shaw Hospital Affiliated to School of Medicine,Zhejiang University,Hangzhou 310016,China)
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2020年第9期1275-1280,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(31970796)
江苏省自然科学基金(BK20160045)
