Establishment Of Research Model On The Selective Activation Of DNA Replication Origins During Zebrafish Early Embryogenesis

DNA replication with high fidelity enables genetic information to be transmitted faithfully and is essential for maintaining the integrity and stability of the genome.In eukaryotes,DNA replication initiates at selectively activated origins,and initiation defects would result in abnormal cell division,apoptosis and cancer,which further causing dysplasia and even death.With the technological development of genome-wide sequencing,ChIP-seq,single-cell sequencing,researchers suggest that primary sequence and chromatin environment contribute to the selective activation of DNA replication origins.However,most of the research work is based on yeast,a unicellular eukaryote,few studies have explored the selective activation of DNA replication origins in higher eukaryotes.Zebrafish begins development with a period of exponential cell expansion via a series of synchronous and rapid embryonic cell cycles without activation of the transcriptome before mid-blastula transition,which provides us with a good in vivo model for studying DNA replication origins.However,due to the lack of high-quality endogenous antibodies in zebrafish,ChIP-seq experiments couldn’t be performed.Therefore,in this study,knocking Flag tag into key genes(such as cdc6,cdtl and mcm5)of pre-RC was performed by optimized the intron-and non-homologous end ligationmediated CRISPR/Cas9 technique.After construction of the transgenic fish,Flag antibody would be used in the ChIP-seq experiment.This lays the foundation for analysis of specific chromatin features of DNA replication origins in combination with functional genomic analysis methods.In this study,gRNAs were designed for four genes,cdc6,cdt1,and mcm5.After analysis,it was found that the gRNAs on the cdc6,cdt1 and mcm5 genomes had relatively good editing effects.The effective gRNA,donor plasmid and Cas9 protein were co-injected into the zebrafish one-cell stage embryo.Through extraction of genomic DNA at 24 hpf and amplification of the target fragment,the insertion of the tag was confirmed by sequencing analysis.This study successfully screened cdt1Flag/Flag and mcm5Flag/Flag transgenic fish that Flag tag was correctly integrated.In addition,it is demonstrated that Flag tagged proteins can mimic the expression of endogenous Cdtl and Mcm5 proteins.In summary,this study successfully constructed cdt1Flag/Flag and mcm5Flag/Flag transgenic fish,which provided a key experimental material for the subsequent study of selective activation of eukaryotic DNA replication origins.