利用CRISPR/Cas9系统制备斑马鱼cbsb敲除模型

Establishment of zebrafish cbsb-knockout model by gene editing with CRISPR/Cas9 system

目的 采用成簇的规律性间隔的短回文重复序列(CRISPR)/Cas9系统对斑马鱼胱硫醚-β-合成酶b基因(cbsb)进行编辑,并制备稳定的斑马鱼cbsb敲除品系。方法 使用ClustalX软件分析斑马鱼cbsb编码蛋白(Cbsb)的保守结构域,利用生物信息学选取向导RNA(gRNA)靶点,体外合成并纯化gRNA和密码子优化的Cas9 mRNA,在刚受精的斑马鱼胚胎(1-cell期)中显微注射混合的gRNA和Cas9 mRNA,提取受精后3 d的斑马鱼幼鱼基因组,通过PCR扩增并测序分析靶点的切割效率,通过基因组扩增和测序筛选并获得cbsb敲除的稳定品系。结果 斑马鱼Cbsb与人、小鼠胱硫醚-β-合成酶(CBS)高度同源。在斑马鱼Cbsb保守结构域的编码序列处设计4个靶点,其中4#靶点具有高效的切割效率,利用该靶点制备并筛选获得多个cbsb敲除斑马鱼,选取-3+13 bps和-228 bps移码突变的两种品系,进一步筛选获得稳定的cbsb敲除品系。结论 利用CRISPR/Cas9系统成功制备了两种斑马鱼cbsb敲除的稳定品系,为进一步在斑马鱼活体中研究Cbsb的功能奠定了基础。

Objective To establish zebrafish cystathionine beta-synthase b gene(cbsb) knockout stable lines by gene editing with the clustered regularly interspersed short palindromic repeats(CRISPR)/Cas9 system.Methods ClustalX software was used to analyze the conserved domain of encode protein of zebrafish cbsb(Cbsb).Bioinformatics analysis was used to choose guide RNA(gRNA) targets.gRNA and codon-optimized Cas9 mRNA were synthesized and purified in vitro,and the mixed gRNA and Cas9 mRNA were microinjected into zebrafish fertilized eggs at 1-cell stage.The genomic DNA of zebrafish larvae 3 days after fertilization(3-dpf) was extracted,and the cleavage efficiency of gRNAs was analyzed by PCR and sequencing.The stable cbsb-knockout strains were screened out by further genomic amplification and sequencing.Results The zebrafish Cbsb was highly homologous to the cystathionine beta-synthase(CBS) of human and mouse.Four targets were designed in the coding sequence of conserved domain of the zebrafish Cbsb,and gRNA 4# had high cleavage efficiency.Multiple cbsb-knockout zebrafish strains were obtained,and two frameshift mutations of-3+13 bps and-228 bps were chose to obtain stable cbsb-knockout lines.Conclusion Two stable cbsb-knockout zebrafish lines have been successfully established by gene editing using CRISPR/Cas9 system,which may be used for further functional study of Cbsb in vivo.