Construction Of Multiplexd SgRNA-CRISPR/Cas9 System And Its Preliminary Application In Genome Imaging

Objective:The nucleus of human cells harbours 46 densely packed chromosomes.Chromosomes are folded into hierarchical domains at different genomic scales,which may be beneficial to the effective assembly and functional organization of the genome.At present,the main techniques for studying chromatin folding are chromatin conformational capture(3C)and imaging techniques,but most of them can only be used for the study of fixed cells.However,the study of chromatin folding in living cells is very important to understand the relationship between spatiotemporal chromatin organization and gene function.In recent years,CRISPR/Cas9 Imaging technology has been proved to be effective in the study of living cell chromatin folding,which can be used for live cell imaging of chromatin sites.In this project,we constructed a multiplexd sgRNA-CRISPR/Cas9 system to initially realize the imaging of chromatin sites and provide a theoretical basis for the study of chromatin folding in living cells.Methods:In this paper,the methods of constructing plasmids include ligation,recombination and Golden-gate clone,in the process,using HiPure Gel Pure Micro Kit(D2111-03)to recycle gel DNA,usingHiPure Plasmid Micro Kit-C(P1001-03C)to extract plasmids.Lipo3000 was used to transfect the cells,and then confocal laser microscope was used for imaging observation.Finally,the imaging data was processed by ImageJ.Results:1.The CRISPR-Cas9 dual light system allow the imaging of repeated genomic sequences.Construction of CRISPR/Cas9 dual light system,which include(CRISPR/Cas9)-(MS2-MCP)and(CRISPR/Cas9)-(PP7-PCP).Although we have successfully achieved the imaging of telomeres and mouse Dusp5 sequences using this system,the imaging of non-repetitive genomic sequences failed because of multiple sgRNAs could not be expressed simultaneously.2.Plasmid-dependent multiplexed sgRNA assembly system.Establish a multiplexed sgRNA assembly system,which was used to assemble multiple sgRNAs into a single plasmid,thus improving the ability of simultaneous expression of multiple sgRNA.3.Imaging of non-repetitive genomic sequences.Using multiplexed sgRNA expression plasmids to achieve the simultaneous expression of 24 kinds of sgRNAs,and then to achieve non-repetitive genomic sequence imaging.conclusion:The establishment of multiplex sgRNA plasmid assembly system can improve the ability of simultaneous expression of multiple sgRNA,thus improving the ability of sgRNA to target genomes,which allows CRISPR imaging system to image non-repetitive genome sequences,expands the application range of CRISPR in the field of genome imaging,and provides a theoretical basis for the study of three-dimensional tissue and dynamics of chromatin.