Preliminary Study On Labeling Of Zebrafish Chromosomes With CRISPR/dCas9 System

The CRISPR system is an immune defense system found in bacteria.The CRISPR/Cas9 system constructed from this has now become a widely used gene editing technology.The CRISPR/dCas9 system is modified on the CRISPR/Cas9 system.The Cas9 protein gene is mutated,so that the Cas9 nucleic acid cleavage function is lost,and it becomes a defective Cas9 protein(deadCas9,dCas9).For example,fusion of fluorescent protein and dCas9 protein can realize the new function of locating the target sequence without cutting the target sequence.Since its establishment,CRISPR/dCas9 technology has been mainly used for the screening and verification of molecular interactions and the research of various gene functions.The application of this system to chromosome markers in living cells can effectively promote the visualization of chromosome dynamics,thereby increasing people's understanding of dynamic processes in the nucleus.However,CRISPR/dCas9 technology is currently less used in chromosome marking,and its application in zebrafish species is rarely reported.Therefore,this thesis conducted a preliminary study on the method of fluorescently labeling zebrafish chromosomes using the CRISPR/dCas9 system.First,we designed an sgRNA sequence that specifically binds to two repetitive regions of zebrafish chromosome 25.The base positions of the repeat regions are 5226 bp-8900 bp and 3746391 bp-37496321 bp.The sgRNA plasmid and dCas9 plasmid were injected into the zebrafish one-cell stage embryos by microinjection,and the embryos were observed by inverted fluorescence microscope at different stages of the embryos.It was found that the plasmid began to express green fluorescent protein in the gastrulation stage,concentrated green fluorescence appeared in the blastomere area of No.1 sgRNA and dCas9 plasmid co-injection groupIn order to observe the green fluorescence of a single cell more clearly,we later used the cell transient transfection method to try to co-transfect the sgRNA plasmid and dCas9 plasmid into the zebrafish ZEM-2S cell line,and optimized the transfection conditions.The study found that when using Neofect transfection reagent,the ratio of transfection reagent to plasmid is 1.5:1,and the concentration ratio of sgRNA and dCas9 is 2:1,the effect of transient transfection 36 h fluorescent labeling is the best.The experiment successfully marked the 5226 bp-8900 bp repeat region at base positions of zebrafish chromosome 25,which laid the foundation for the subsequent flow sorting of chromosomes and the construction of zebrafish-specific chromosomal libraries.