Construction Of (CRISPR/Cas)-based Imaging System And Simulation Of The Spatial Position Of Chromosome In A HEK293T Cell's Nuclei

Human genomics has experienced two major waves of development.The first is the Human Genome Project,whose main purpose is to determine the nucleotide sequence of human chromosomes and to plot the genome of the human genome.The second is the "Encyclopedia of DNA Elements" program,and the main purpose of the program is to identify,analyze,interpret and annotate the functional elements of the human genome.After we got the sequence of the human genome and knew the role of functional components,naturally we want to know the interaction between the functional components of the gene and their impact on the genes of great interest,which also promote the development of the human genomics.The third wave of the arrival of 3D genomics studies the real existence of the three-dimensional structure of the gene expression and its regulation in the nucleus,and then objectively and systematically responses to the genome and its regulation on the biological phenotype.We are now experiencing this wave.Recently,most of the researches on 3D genetics have been studied by chromosome conformation capture techniques such as 3C,4C,5C,Ch IA-PET and Hi-C,and the these data are analyzed to study the interaction between gene sequences.A direct observation of the interaction can be done by imaging.In this paper,a simple,easy-to-operate,multi-color in vivo imaging technique was established by combining the CRISPR/Cas system with the MS2-MCP system from MS2 phage and the(box B)-(N protein)system of ? phage respectively.The spatial position of some chromosome in HEK293 T cells was observed by this method,and the spatial relative position of chromosome in the nucleus was finally simulated.(1)Construction of system(CRISPR/Cas)-(MS2-MCP)and system(CRISPR/Cas)-((box B)-(N protein)).The tetraloop and stem-loop2 of sg RNA were added with the same sequence MS2/(box B),and the protein MCP/(N protein)recruited by MS2/(box B)was fused with a nuclear localization signal,followed by overlapping PCR with e GFP/m Cherry for fusion expression.(2)Inspection of system(CRISPR/Cas)-(MS2-MCP)and system(CRISPR/Cas)-((box B)-(N protein)).The targets of g RNAs of the two systems were set to the same sequence,namely MUC4-E3 gene.Then the plasmids of the two systems were transfected into HEK293 T cells.It was observed that the green fluorescent spots emitted by the system(CRISPR/Cas)-(MS2-MCP)appeared in the same position as the red fluorescent spots emitted by the(CRISPR/Cas)-((box B)-(N protein)),and the feasibility of the two systems was verified.The system(CRISPR/Cas)-((box B)-(N protein))was verified by FISH,and the sequence targeted by the FISH probe was also the MUC4-E3 gene.The plasmid of(CRISPR/Cas)-((box B)-(N protein))was transfected into HEK293 T cells,then FISH-related operation was carried out,and the FISH probe with green fluorescent was introduced into the cells.The green fluorescence emitted by the probe and the red fluorescence emitted by system(CRISPR/Cas)-((box B)-(N protein))can overlap,which can further verify the feasibility of the two system.(3)Look for targets of g RNA and design g RNA.We use the suffix array algorithm to find tandem repeat sequences that appear only on a chromosome within the human genome so that fluorescence signals emitted by the two systems targeting this sequence can be aggregated and amplified.The repetitive sequences on chromosome1,chromosome2,chromosome19 and chromosome22 were selected as the target sequences,and the g RNA was designed according to the related requirements and methods and then connected to the constructed system.(4)Observe the spatial position of the chromosomes in HEK293 T cells and simulate their spatial positions in the nucleus.The four chromosomes that were targeted in HEK293 T cells were observed by confocal microscopy,and the cells were reconstructed in three-dimension space,so that the chromosomes in the nucleus were targeted and their spatial positions were known.We could get the coordinates of each chromosome in space,and eventually simulate the spatial position of four chromosomes in the nucleus.System(CRISPR/Cas)-(MS2-MCP)and system(CRISPR/Cas)-((box B)-(N protein))based on CRISPR/Cas were successfully constructed by above studies.Four chromosomes in HEK293 T cells were successfully observed and their spatial positions in HEK293 T cells could be sure and be simulated.In order to further study the other chromosomes and even the spatial relative positions of other chromosome in a nuclei.This method provides a new ideaand a new perspective,and provides a theoretical basis for the 3D genomics research.