Study On Induced Mutation From CCR5 Wild Type To CCR5?32 In Lymphoid Cells By Using CRISPR/Cas Gene Editing System

Human immunodeficiency virus(HIV)is the pathogen of AIDS,HIV type1(HIV-1)is the main type that causing global pandemic.HIV-1,as an infective virus,has strong replication ability and destructive power,which forms an enormous threat to the safety of people's life and causes a great panic in the world.According to the different coreceptor usage in the process of entry fusion,HIV-1 can be divided into X4-tropic viruses and R5-tropic viruses.X4-tropic viruses use CXCR4 as a coreceptor and always appear at the late stage of HIV-1 infection,and R5-tropic viruses use CCR5 as a coreceptor,and are the main strains in the new HIV-1 infected patients.It is reported that only one patient,Berlin patient,suffering from HIV/AIDS and acute myeloid leukemia,has been cured due to allogeneic hematopoietic stem cell transplant.The donor has a homozygous CCR5?32 mutation in the CCR5 gene.This therapy is unique,but not likely to be widely used,because individuals who have homozygous CCR5?32 mutation are less in the population.To disrupt HIV-1entry coreceptors CCR5 or CXCR4 by using gene-editing system has raised possibility of preventing virus into the cell,but the risk of deleting these molecules is unknown.In this study,we attempt to induce CCR5 gene into CCR5?32 mutation using CRISPR / Cas technology.Fistly,we designed a pair of single-guide RNA(sgRNA)on both sides of the 32 bp and transfected the CRISPR plasmid containing the two sgRNA into Jurkat cells or primary CD4+ cells by lentivirus packaging system.The results of T7 endonuclease 1(T7E1)enzyme digestion and DNA sequencing revealed that CRISPR/Cas system successfully induced CCR5?32 mutation in these two types of cells.It was up to 75 percent cells that was found to be mutated into CCR5?32deletion in Jurkat cells,but only 33 percent in primary CD4+ cell.This difference may be due to the difficulty in transfection of primary cells,which results in less transfected cells containing two kinds of sgRNA.Then,we obtained a number of monoclones in Jurkat cells by using limited dilution method,and selected five ofthem to further analyze their CCR5 genetype,we found that one of them was CCR5?32 homozygous mutation.It is the first time that using CRISPR/Cas system acquire homozygous CCR5?32 mutation monoclones in Jurkat cell and introduce CCR5?32 mutation in primary CD4 + cells,and no apparant off-target effects were found.This study provides a potential clinical application for HIV/AIDS cure in the future.