Construction Of A Dual-color Multiplex CRISPR/sgRNA System And Its Preliminary Application In Zebrafish Chromosomal Imaging

Chromosomes are the carriers of genetic information in eukaryotes.The close relationship between their structure and function has prompted people to explore the three-dimensional structure of chromatin at both time and space scales.Although various chromosome imaging methods have been developed,such as zinc finger proteins,fluorescence in situ hybridization,and Hi-C,these methods all have certain limitations.The emergence of the clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins(CRISPR/Cas)technology has opened up a new field for chromosome imaging techniques.Currently,various chromosome imaging systems based on CRISPR/Cas technology have been developed,such as CRISPR-Sirius and dCas9-Sun Tag.The advantages of these systems are that DNA denaturation and other operations are not required during the imaging process,which greatly maintains the normal life activities of chromosomes in living cells.Zebrafish is one of the internationally recognized model species in modern biology,with the advantages of easy genetic control and high genetic similarity to humans,making it an ideal model for studying human disease-related genes.The transparency of zebrafish embryos provides important materials for exploring organ development,neural regulation,lineage tracing,and other aspects,and also has great significance for the study of chromosome structure and dynamics.This study utilized overlapping PCR technology to construct a dual-color fluorescence imaging system based on CRISPR/dCas9 and MS2/MCP,PP7/PCP.The experimental results demonstrated that the two imaging systems can specifically target highly repetitive sequences on the chromosomes of zebrafish Zem-2S cells.A polycistronic t RNA-sgRNA plasmid expression vector was established based on Golden-gate cloning technology,combined with the MS2/MCP,PP7/PCP dual-color fluorescence imaging system to achieve imaging of moderately repetitive sequences on chromosomes 24 and 25 in live zebrafish Zem-2S cells.In addition,the two imaging systems can simultaneously label two pairs of homologous chromosomes in a single live zebrafish cell,and monitored the position and distance changes of the two pairs of homologous chromosomes and track the spatial position changes of chromosomes in real-time.This study also examined the toxicity of the polycistronic t RNA-sgRNA in the imaging system on cells,and found that transient transfection of the polycistronic t RNA-sgRNA vector had lower toxicity to cells than transient transfection of a single sgRNA vector when equal amounts of sgRNA were transfected.There are few reports on imaging zebrafish chromosomes using CRISPR/dCas9 technology,and the results of this study can lay an experimental foundation for the spatial imaging study of zebrafish chromosomes.