Effect Of Lentivirus-based CRISPR System On Human Chromosome Variations

In recent years,as life science research has advanced,gene-editing techniques have emerged,and have been used to treat a variety of diseases.CRISPR(clustered regularly interspaced short palindromic repeats)gene editing techniques have been widely studied for its unique advantages,but like other gene editing techniques,off-target effects are unavoidable.The effect of off-target may cause other diseases such as cancer,which limits the use of gene editing techniques in the clinical treatment of various diseases.Double-strand breaks(DSBs)can occur in the target region,thereby activating DNA damage repair mechanisms to induce insertions or deletions.Copy number variations(CNVs)is the addition or deletion of gene copy number on human chromosomes.It has scarcely been reported whether using CRISPR system to edit human genomes will result in copy number variations.This study attempted to edit the CFTR(Cystic Fibrosis Transmembrane Conductance Regulator)locus of human genome through a lentiviral vector-mediated CRISPR/cas9 system and detect copy number variations of single cell by next-generation sequencing.The main research results are as follows:(1)Construction of the lentiCRISPRv2-CFTR vector.Select the cutting sites and design sgRNA.The sgRNA with sticky end restriction site was synthesized by double strand annealing and it was ligated into the lentivirus transport plasmid lentiCRISPRv2 by enzyme-digested and enzyme-linked reaction.LentiCRISPRv2 plasmid has a puromycin resistance gene which can be used for the screening of subsequent stable cell lines.The lentiCRISPRv2-CFTR vector was successfully constructed.(2)Selection of target cells for editing.Primers were designed to amplify the sequence near target sites of the cell genome by PCR and then the PCR product was ligated to a T-vector.Sequencing verifies the consistency of sequences near the target sites of two sister chromatids of cells,and selected cell with consistent sequences near target sites of two sister chromatids as target cells for editing.The target cell for CRISPR/cas9 editing system is successfully selected.(3)Lentivirus packaging and preparation of stable cell lines.The lentivirus packaging plasmids and recombinant transporter plasmid was co-transfected into lentiX 293T cells through calcium phosphate transfection and produced lentivirus particles with sgRNA by cell culture.Lentivirus mediated sgRNA into target cells and integration its own genome into the cell genome,and the stable cell lines were successfully obtained by puromycin screening.(4)Stable single cell genome amplification and next-generation sequencing.Stable single cells were picked through manual cell picking,and their genomes were amplified with a single cell whole genome amplification kit.The sequence near target sites of single cell genome was amplified by PCR and then ligated to T-vector to verify whether the target site was edited through sequencing.Next-generation sequencing of the edited single-cell genome and use CNVkit to analyze whether CRISPR editing will result in copy number variation.Next-generation sequencing result of single-cell whole genomes based on multiple displacement amplification(MDA)indicate poor uniformity of genome,which results in inaccurate CNVs analysis.Although this study failed to accurately detect CNVs,it lays the foundation for CNVs analysis of single-cell.In subsequent experiments,we will use single-cell amplification kits of other principles to amplify single-cell genomes and attempt to analyze CNVs.