Establishment of zebrafish cbsb-knockout model by gene editing with CRISPR/Cas9 system
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.
Cystathionine beta-synthase bHydrogen sulfideGene knockoutZebrafishClustered regularly inter-spersed short palindromic repeats(CRISPR)/Cas9
万方数据
维普
