Plasmid Delivery CRISPR System And Cell Chromosome Copy Number Variations

With the birth of new generation of gene editing technology CRISPR system,gene editing has entered a modern era.In the CRISPR-Cas9 system,Cas9 generates double-stranded breaks(DSBs)at target sites under the guidance of the guide RNA,activating DNA repair pathway to achieve gene editing.The process of DNA repair can easily lead to insertion or deletion mutations,and even cause diseases such as cancer.One of the mechanisms of copy number variants(CNVs)is the chromosome repair caused by DSBs,and no studies have shown whether the off-target effect of the CRISPR-Cas9 system triggers genome-wide copy number variation.The purpose of this study was to use a plasmid delivery CRISPR system to edit the genome of cells and to detect genome-wide copy number variation using single cell genome sequencing.The main research results are as follows:(1)Select a human cell line that is suitable for gene editing.Transient tests of three kinds of human cell lines MRC-5,NCI-N87,HCT-116 were performed by calcium phosphate transfection,liposome transfection and electroporation.At the meantime,sequence of target loci on two homologous chromosomes was verified.Combining the above factors,HCT-116 cells which are easy to transfect and have the same sequence of cleavage sites on homologous chromosomes were successfully selected as experimental cell lines.(2)Construction of PX459-CFTR plasmid vector.Two sgRNAwere designed for the cleavage site on the CFTR gene sequence.Direct double-stranded annealing and then connected to the PX459 vector(the vector carrying puromycin gene,which can be applied to subsequent drug screening)and successfully constructs the PX459-CFTR plasmid vector.(3)Cell transfection and screening of positive cells.PX459-CFTR plasmid vector was transferred into HCT-116 cells by liposome transfection,and puromycin was used to screen the transfected cells for three rounds of drugs.Finally,the positive cells were successfully obtained.(4)Single-cell genome amplification and high-throughput sequencing of positive cells.Single positive cell was picked up and the genome amplification was performed using single-cell amplification kits.Editing sites were amplified to verify the success of gene editing.Five successfully edited single-cell genomes were obtained,high-throughput sequenced and copy number variation were analyzed using CNVKit.It turns out that the amplification of the cell genome with the principle of MDA was not suitable for the study of copy number variation.In this experiment,the CRISPR-Cas9 system was successfully used to edit the mammalian cells,and the method of MDA was used to enlarge the single-cell genome.High-throughput sequencing results show that the proposed method can cause genomic coverage unevenly,resulting in the deviation of sequencing results and the accurate quantification of genomic copies.The results of this experiment shows that the MDA method is not suitable for the study of copy number variation,and subsequent attempts should be made to use other amplification methods or combine other detection methods such as aCGH for copy number analysis.