Efficient Genome Editing In Human Pluripotent Stem Cells Using CRISPR/Cas9 Technology

Human pluripotent stem cells(hPSCs),including embryonic stem cells(hESCs)and induced pluripotent stem cells(hiPSCs),have unlimited self-renewing capacity and the potential to generate all adult cell types.Therefore,hPSCs provide an unprecedented powerful platform for human disease research,exploring embryonic development,drug screening,and regenerative medicine.To investigate the gene function via hPSCs,it is necessary to manipulate their genomes in a precise and efficient manner.However,gene targeting in hPSCs has been extremely difficult.Recently,CRISPR/Cas9 system has been applied to genome engineering,which greatly increases the efficiency and diversity of genome editing in hPSCs.In this study,we have developed a genome-editing platform in hPSCs using lentiviral Cas9 to establish gene knockout models rapidly and efficiently.We established PRDM1 and VASA mutant hPSCs associated with germ cell development,as well as hPSCs with mutantation in DBY,a gene which is located on AZFa region,associated with nonobstructive azoospermia(NOA).In addition,we successfully established hPSCs containing large deletion of genomic DNA fragment.We further screened and analyzed the function of genes that are related to germ cell development.